viral drivers of honey bee losses the dwv-clade and the ... · →honey bee health is influenced by...
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Viral drivers of honey bee losses ‒
the DWV-clade and the AKI-complex
© Schäfer
Marc Oliver Schäfer
OIE Symposium on Emerging
Infectious Agents in Honey Bees &
OIE-Listed diseases
02.10.2017
Honey bee colony lossesEstimated annual honey bee colony mortality in Europe
Colony mortality rates [%] in all participating Member States of the
European Union, recorded throughout the EPILOBEE study
© European Commission, ANSES, EURL (modified)
Mayor drivers of losses (Jacques et al. 2017):
• Beekeeper background and apicultural practices
EPILOBEE 2012/13 EPILOBEE 2013/14
Honey bee colony losses
Summary of total overwintering colony losses in the US (The acceptable winter
loss rate shows the average of acceptable yearly colony losses declared by
survey participants in each year)
Estimated honey bee colony mortality in the US
© beeinformed.org
The top five reasons (vanEngelsdorp et al. 2012):
• Starvation
• Weak colonies in the fall
• Poor wintering conditions
• Poor queens
• Varroa mites
Estimated percentage of
colonies lost due to each
commonly self-reported
colony health risk factors
in season 2016-2017
(Kulhanek et al. 2017).
© Kulhanek et al. 2017
Honey bee colony lossesEstimated honey bee overwintering colony mortality in Germany
Results of the German bee monitoring project (‘DeBiMo’) in comparison with a
written survey (‘MAYEN’ = passive surveillance) and the results of the pan-
European epidemiological study ‘EPILOBEE’
0
5
10
15
20
25
2004/05 2005/06 2006/07 2007/08 2008/09 2009/10 2010/11 2011/12 2012/13 2013/14 2014/15 2015/16 2016/17
written survey "MAYEN" monitored colonies "DeBiMo" all colonies "DeBiMo" "EPILOBEE" Surveyed winter
Mort
ality
[%]
The identified factors involved (Genersch et al. 2010):
• High V. destructor infestation levels
• Clinically relevant DWV infections in autumn
• ABPV infections in autumn
• Old queens
• Relative colony weakness before overwintering
Factors that influence honey bee health
Beekeeping
practice
Quantity & quality
of the nutrition
© Kathy Keatley Garvey
© ALP © Ritter
© Zach Huang
Diseases of the honey bee
© Fries
Weather / climate Vitality & diversity
of the honey bees
Pesticides © claudiaspahr.com
© Schäfer
© Schäfer
Bacteria (e.g. Foulbrood)
Fungi (e.g. Chalkbrood)
Endoparasites (e.g. Nosema apis & N. ceranae)
Parasitoids (e.g. A. borealis)
Viruses(e.g. DWV, SBV)
Other pests(e.g. Aethina tumida & V. velutina)
Ectoparasites(e.g. Varroa & Tropilaelaps spp.)
Diseases of the honey bee
© Fries
© Zach Huang
© Ritter
© Rosenkranz
© Jean Haxaire
© Aronstein
© USDR-ARS
© Schäfer
© PlosOne
Viruses associated with honey bees
© Francis 2012 © Francis 2012
The AKI-complex
The DWV-clade
AKI-complex world distribution
© Elsevier / Journal of Invertebrate Pathology 103, de Miranda et al. 2010 (modified)
West African Republic of Benin (Amakpe et al. 2015)
Argentina (Molineri et al. 2017)
DWV world distribution
© IBRA / Bee World 86, Ellis and Munn 2005
© Science 351, 10.1126/science.aac9976, Wilfert et al. 2016 (modified)
Wilfert et al. (2016) have shown that DWV is
globally distributed, (A) driven by trade and
movement of honey bee colonies, and (B) is
following the spread-pattern of V. destructor
However, a recent study showed the
absence of DWV in Australia
(Roberts et al. 2017)
© Roberts JMK, Anderson DL and Durr PA 2017
© Science 351, 10.1126/science.aac9976, Wilfert et al. 2016 (modified)
© Elsevier / Journal of Invertebrate Pathology 92, Chen et al. 2006 (modified)
Transmission of viruses in honey bees
© Schäfer© Schäfer
© Gudrun Koeniger
Transmission of viruses through
Varroa destructorVarroa destructor is transmitting several viruses:o DWV (Bowen-Walker et al. 1999)o ABPV (Ball 1983)o KBV (Chen et al. 2004, Shen et al. 2005)o IAPV (de Prisco et al. 2011)
Varroa destructor is a biological vector for DWV (Ongus et al. 2004, Yue and Genersch 2005)
egg
1
egg
2
egg
3
egg
4
egg
5
mating
Mother mite with
two daughter mites
leaving the worker
brood cell
© Elsevier / Journal of Invertebrate Pathology 103, Rosenkranz et al. 2010 (modified)
Protonymph
Deutonymph
Adult maleAdult female
© Schäfer
Varroa destructor
+
AKI-complex
DWV-clade
BQCV
CBPV
SBV Other
viruses ?
© Elsevier / Current Opinion in Insect Science 8,
McMenamin and Genersch 2015 (modified)
Transmission of viruses through
Varroa destructor
Clinical symptoms of “paralysis”
Death of lethally infected bees is preceded by a rapidly progressing paralysis,
including trembling, inability to fly and the gradual darkening and loss of hair from
the thorax and abdomen (ABPV & IAPV), symptoms usually associated with CBPV.
Severe ABPV & KBV infection causes a sharp decline in the adult bee population.
Photos courtesy of The Animal and Plant Health Agency (APHA), © Crown Copyright
Clinical symptoms of IAPV infection
© PLoS ONE 8(9): e73429; Boncristiani et al. 2013
Clinical symptoms of DWV infection
Pupal death • emergence of non-viable, malformed bees • discolouring • healthy
looking bees suffering from ataxia and disorientation (Yang and Cox-Foster 2007).
Such bees are non-viable and die within 67 hours after emerging (Möckel et al. 2011).
© Rosenkranz© Schäfer
© Schäfer © Schäfer
© ALP
Viruses and Varroa destructor
DWV has changed dramatically in prevalence, load and strain diversity after the
new arrival of V. destructor into former mite-free honey bee populations in Hawaii
= Varroa free
= Varroa infested
Hawaii 2009
Viral load, prevalence and genetic
diversity of DWV across the four
main islands that have been
exposed to Varroa destructor for
different periods of time
© Science 336, 10.1126/science.1220941, Martin et al. 2012 (modified)
© Rosenkranz
Viruses and Varroa destructor
© Roberts JMK, Anderson DL and Durr PA 2017
Viruses and Varroa destructor
N=2.540
N=1.034
N=192
0
5
10
15
20
25
30
kein DWV / kein ABPV DWV oder ABPV DWV und ABPV
V.destructor
pe
r 1
00
be
es
[%]
B*
C*
A*
no ABPV/DWV ABPV or DWV ABPV and DWV
Results of the German bee monitoring (data from 2009-2015) show that the
higher the colony infestation level with V. destructor, the higher is the
chance of infections with ABPV and/or DWV
* Different letters indicate a significant difference (H-Test (Kruskal-Wallis); P<0,001)
Viruses and colony losses
If ABPV or DWV are detected in a colony in autumn, the higher is the
chance that the colony will not survive the following winter
N=2.736
N=968
0
5
10
15
20
25
2009 - 2016
Ove
rwin
teri
ng
mo
rtal
ity
[%]
DWV nicht im Volk gefunden
DWV im Volk gefunden
***
*** Chi-Quadrat; P<0,0001
Colony not infected with DWV
Colony infected with DWV
N=3.288
N=416
0
5
10
15
20
25
2009 - 2016
Ove
rwin
teri
ng
mo
rtal
ity
[%]
ABPV nicht im Volk gefunden
ABPV im Volk gefunden
*
* Chi-Quadrat; P<0,01
Colony not infected with ABPV
Colony infected with ABPV
Viruses and colony losses
© SCIENCE / 10.1126/science.1146498, Cox-Foster et al. 2007 (modified)
© SCIENCE / 10.1126/science.1146498, Cox-Foster et al. 2007 (modified)
CCD has resulted in a loss of
50 to 90% of colonies in
affected beekeeping
operations across the US
IAPV was strongly correlated with
CCD (Cox-Foster et al. 2007)
Honey bee virus diagnostic
Enzyme-Linked ImmunoSorbent Assay (ELISA)
ELISA is a complex multistep assay involving sensitive enzymatic reporters.
There are many opportunities for assay failure.
Polymerase Chain Reaction (PCR) is the most commonly used technique
Gel-based detection Fluorescence-based detection
Nested/semi-nested PCR
classical PCRReal-time PCR without probes (intercalating dyes)
Real-time PCR with probes (TaqMan, FRET, Beacon)
• Highly sensitive and specific
• moderate costs for consumables and PCR cycler
• Sensitive and specific
• moderate costs for consumables and PCR cycler
• Sensitive
• Time-saving
• Reduced risk of cross-contaminations
• High-throughput analyses
• Semi-/quantitative results
• Sensitive and high specific
• Time-saving
• minimal risk of cross-contaminations
• High-throughput analyses
• Semi-/quantitative results
• Time-consuming
• Qualitative results
• Very high risk of cross-contaminations
• Confirmation of specificity by fragment size
• Time-consuming
• Qualitative results
• High risk of cross-contaminations
• Confirmation of specificity by fragment size
• Confirmation of specificity by melting curve analysis
• Cost for real-time PCR cycler
• Reduced diagnostic sensitivity based on additional probe
• Cost for probes and real-time PCR cycler
P
r o
s
C
o
n
s
0
20
40
60
80
100
ABPV BQCV CBPV DWV KBV SBV Varroa
0
20
40
60
80
100
ABPV BQCV CBPV DWV KBV SBV Varroa
Occurr
ence
[%]
Occurr
ence
[%]
Honey bee virus diagnostic
Honey bee virus diagnostic
The most common bee viruses use their own RNA-dependent RNA polymerases to replicate.
These enzymes lack the proof-reading capability seen in their DNA counterparts, and this leads
to high error rates. The resulting genotypic variants give the virus a remarkable evolutionary
rate and the mixed population of mutant viral genotypes is referred to as a quasispecies.
For DWV it is already accepted that it exists as a quasispecies (Martin et al. 2012, Mordecai et
al. 2016), that is also called the DWV-clade (de Miranda and Genersch 2010).
© European Communities, Virology and the Honey Bee, 2008
Honey bee virus diagnostic
DWV-clade diagnosticConventional PCR targeting multiple
regions of the DWV genome
© Elsevier / The Veterinary Journal 169, Genersch 2005 (modified)
© McMahon et al. 2017, Proc. R. Soc. B 283, the Royal Society (modified)
Real–time SYBR Green PCR targeting
multiple regions of the DWV genome
Real–time TaqMan® PCR
© Chen et al. 2005, Applied and Environmental Microbiology 71 (modified)
© Forsgren et al. 2009, Springer / Exp. Appl. Acarol. 47 (modified)
© Elsevier / The Journal of Invertebrate Pathology 91, Chantawannakul et al. 2006 (modified)
With the use of (next-generation)
sequencing the DWV quasispecies will be
further investigated. Thus, Mordecai et
al. (2016) have shown that the DWV
species complex is made up of three
master variants: DWV Type A, B and C
AKI-complex diagnosticReal–time SYBR Green PCR targeting multiple regions of the
IAPV genome
Real–time TaqMan® PCR
AKI Primers
The design of primers
that allow the detection
of ABPV, KBV and IAPV in
a single assay reduce cost
& time. Only AKI-positive
samples will be combined
with specific PCRs.
© Elsevier / The Journal of Invertebrate Pathology 91, Chantawannakul et al. 2006 (modified)
© PLoS ONE 7 e30641, Evison et al. 2012 (modified)
© American Society for Microbiology / Journal of Virology 82, Palacios et al. 2008 (modified)
© Francis and Kryger 2012, Journal of Apicultural Science 56 (modified)
Conclusions
→ Varroosis seems to be a main cause for worldwide honey bee
colony losses
→ Varroosis = infestation with V. destructor plus infection with
viruses that are associated with the mite
→ Honey bee health is influenced by multiple factors, diseases play
a major role
→ Most of honey bee viruses are RNA-based and have a quasispecies
nature. For DWV, its quasispecies nature is already accepted
→ Viruses of the DWV clade and the AKI-complex are spread
worldwide. Where there are bees, there are viruses
→ Detection of honey bee viruses is currently based on RT-PCR
methods and new developments will improve future diagnostics
→ The combination of clinical symptoms and the diagnosis of distinct
virus-combinations and their titers may help explaining bee-losses
OIE-listed diseases of the honey bee
American
foulbrood
Varroosis
Acarapisosis
Small hive beetle
infestation
Tropilaelaps
infestation
European
foulbrood
© Forsgren
© Schäfer
© Schäfer
© Schäfer © Schäfer
© Schäfer
© Schäfer
OIE-listed diseases of the honey bee
American
foulbroodAcarapisosis
Small hive beetle
infestationTropilaelaps
infestation
European
foulbrood
© Forsgren
© Schäfer
© Schäfer
© Schäfer © Schäfer
© Schäfer
© Schäfer
Varroosis and
associated viruses© ALP
Dr. Marc Schäfer
National Reference Laboratory for Bee Diseases
Institut of Infectology (IMED)
Friedrich-Loeffler-Institut │FLI│Federal Institute for Animal Health
Südufer 10 | 17493 Greifswald - Insel Riems
GERMANY; Tel: +49 38351 7 1246
http://www.fli.de/index.php?id=251
Thank you!