Pests and Pathogens Varroa and Viruses Orlando Yañez Peter Neumann Institute of Bee Health University of Bern

What is causing bees to decline?

• GM crops • Pesticides • Poor beekeeping

management • Pollution • Electromagnetic

waves • UFOs • Parasites and

diseases

Varroa destructor (Acari: Varroidae)

> Varroa destructor is a new parasite of European honey bees.

> Since its introduction, it is the greatest threat for apiculture.

> Without treatment, most colonies would collapse within a 2–3 year period.

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Photo by IBRA

Interspecific invasion from Apis cerana to Apis mellifera

> Spread almost worldwide within a relatively short time period.

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As a new parasite, a balanced host-parasite relationship is lacking.

host–parasite adaptation in the original host Apis cerana

> Hygienic traits: Grooming behavior Uncapping and

removal Entombing

> Population dynamic

aspects (Reproduction only in drones)

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A. mellifera A. cerana

Mite biology

> Varroa is closely linked to its honey bee host and lacks a free living stage.

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Reproductive phase

Phoretic phase

Transportation and spread

Varroa as virus vector

> It was though, that parasitism by Varroa was the solely cause for adult bee malformations.

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Bowen-Walker et al 1999

Varroa as virus vector

> Adult bee malformations was associated to a honey bee virus.

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Bowen-Walker et al 1999

Non-deformed bees

Mites and deformed bees

Association between Varroa and viruses at colony level

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Survived colony

Collapsed colony

Varroa-virus associated with the collapse of honeybee colonies (Carreck et al 2010)

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• Symptoms • sacbrood • paralysis • black queen cells • deformed wings

• Serology • injection assays - propagation • inapparent picorna-like viruses • ~18 viruses • ~10 picorna-like viruses

Bailey & Ball - Rothamsted Research (UK)

Honey Bee Viruses: Characterization

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• Symptoms • sacbrood

• deformed wings

• paralysis

• black queen cells

Bailey & Ball - Rothamsted Research (UK)

Characterization

VIRUS •Sacbrood virus

•Thai Sacbrood •Chinese Sacbrood

•Deformed wing virus • Kakugo • Varroa destructor-1

• Slow bee paralysis virus •Acute bee paralysis virus

• Kashmir bee • Israeli bee paralysis

• Black queen cell virus • Chronic bee paralysis virus • Cloudy wing virus • Bee virus

• bee virus X • bee virus Y

• Arkansas bee virus • Macula-like virus • Apis iridescent virus • Filamentous virus

FAMILY

Iflavirus

Picorna-like

Dicistrovirus

(New virus family) ? ?

? Maculavirus

Iridovirus Baculo/Ascovirus

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Characterization Picornavirales

> “small” > (+) Single-Strand RNA virus, > Nonenveloped > Icosahedral capsid, 30 nm

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Iflavirus 9~10 kb

5’ 3’ VP3 AAAAAAAA LP IRES

VP1 VP2 helicase VPg 3C-pro RdRp

Dicistrovirus 9~9.5 kb

5’ 3’ AAAAAAAA IRES

helicase VPg 3C-pro RdRp VP2 VP3 VP1 IRES

SBV DWV/VDV-1 SBPV ABPV/KBV/IAPV BQCV CBPV

Characterization Picornavirales

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X

Horizontal Transmission (Same Generation) By feeding, cleaning, robbering, drifting...

Pollen, Larval food, feces

Vertical transmission (next Generation)

No Symptoms (= latent)

from: Chen et al. 2006

Sperm

Egg

Virus transmission routes

In general, low virulence useful for the host and for the viruses = No problems for bees and beekeepers

Varroa has changed the rules of the game

because Varroa added a new transmission route

Viruses and Varroa destructor

1. The mite injects viruses directly into the bee haemolymph

Acute Bee Paralysis Virus ABPV

Slow Paralysis Virus SPV

Deformed Wing Virus DWV

Sacbrood Virus SBV

Kashmir Bee Virus KBV

Israeli Acute Paralyis Virus IAPV

Black Queen Cell Virus BQCV

100 nm

Red: Transmitted by Varroa destructor

Elimination of the first defense mechanisms (e.g. intestinal lamina)

Viruses and Varroa destructor

% brood with KBV

The more mites in a cell, the more likely a virus is transmitted (4 mites ~ 100% at KBV)

Chen et al. 2006

1. The mite injects viruses directly into the bee haemolymph

Viruses and Varroa destructor

2. Mite suppress the immune response of the bee

i.e. Mosquito suppress blood clotting

Viruses and Varroa destructor

1. The mite injects viruses directly into the bee haemolymph

Suppressed immune response = increased susceptibility to viruses?

2. Mite suppress the immune response of the bee

Viruses and Varroa destructor

1. The mite injects viruses directly into the bee haemolymph

3. Virus replication in Varroa can promote virulence

2. Mite suppress the immune response of the bee

Viruses and Varroa destructor

1. The mite injects viruses directly into the bee haemolymph

Viruses and Varroa destructor

Bees showed only crippled wings, when the virus was able to successfully replicate in the mite

DWV: Virus-function analysis in mites from bees with and without symptoms

clear evidence for viral replication

Virus + RNA

Virus - RNA

Yue & Genersch 2005

3. Virus replication in Varroa can promote virulence

2. Mite suppress the immune response of the bee

Viruses and Varroa destructor

1. The mite injects viruses directly into the bee haemolymph

Capsid Proteins Non-Structural Proteins

Helicase Protease Replicase

Genome (+ RNA)

However, no specific drugs are available for the bee viruses.

Control

For viruses of humans (influenza, HIV), there are medications (antivirals)

Prevent penetration into the host cell or stop replication in the host cell (i.e. blocking the protease enzymes)

Currently, RNA interference technologies are being developed against honey bee viruses and Varroa (2020?)

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Control Small Interfering RNA (siRNA)

> RNA interference is a vital part of the immune response to viruses. > Andrew Fire and Craig C. Mello - Nobel Prize (2006) for their work

on RNA interference.

Processing of dsRNA Transfer to Effector Activated Effector

Hammond (2005)

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Control Small Interfering RNA (siRNA)

siRNA Effector Attach to target mRNA

Foreign mRNA

Cleavage of Foreign mRNA

> RNA interference is a vital part of the immune response to viruses. > Andrew Fire and Craig C. Mello - Nobel Prize (2006) for their work

on RNA interference. Hammond (2005)

> Protection of the honey bee against Deformed wing virus (DWV)

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Control Small Interfering RNA (siRNA)

Desai et al 2012

Bees feed with DWV

Bees feed with dsRNA + DWV

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Control Small Interfering RNA (siRNA)

> Protection of the honey bee against Israelí acute paralysis virus (IAPV)

> RemebeeTM by Beeologics > Laboratory assays.

Maori et al 2009

Control Large-Scale Field Application of RNAi Technology against IAPV

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Honey production

Hunter et al 2010

> Under FDA review for use in practice by feeding in syrup to hives.

So, are we completely helpless against bee viruses until RNAi technology is commercially available?

Minimize transmission between colonies (drifting, robbery)

Prevention

A strict control of Varroa and other diseases; stress prevention

Minimize transmission between colonies (drifting, robbery)

Prevention

Bees use propolis, which has antiviral properties

Support bees immune system?

A strict control of Varroa and other diseases; stress prevention

Minimize transmission between colonies (drifting, robbery)

Prevention

Breeding Virus-tolerant bees?

Support bees immune system?

More stringent criteria for the control of Varroa and other diseases; stress prevention

Minimize transmission (drifting, robbery)

Prevention

1. Bee viruses are widespread, certainly underestimated because latent infections without symptoms (= healthy colonies)

Summary

© Ellis & Munn

© Ellis & Munn

ABPV

SPV

DWV

SBV

KBV

BQCV

CPV

CWV

2. Varroa destructor as a virus vector changes the rules of the game

1. Bee viruses are widespread, certainly underestimated because latent infections without symptoms (= healthy colonies)

Summary

3. Activation of viruses by Varroa destructor

2. Varroa destructor as a virus vector changes the rules of the game

1. Bee viruses are widespread, certainly underestimated because latent infections without symptoms (= healthy colonies)

Summary

4. No available remedies for viruses, certainly not at short term period.

Antivirals

3. Activation of viruses by Varroa destructor

2. Varroa destructor as a virus vector changes the rules of the game

1. Bee viruses are widespread, certainly underestimated because latent infections without symptoms (= healthy colonies)

Summary

5. Prevention: Minimizing transmission, strict control Varroa + other diseases, breeding? Propolis?

4. No patent remedies for viruses, certainly not in the long term control

3. Activation of viruses by Varroa destructor

2. Varroa destructor as a virus vector changes the rules of the game

1. Bee viruses are widespread, certainly underestimated because latent infections without symptoms (= healthy colonies)

Summary

1. Bee viruses are widespread, certainly underestimated because latent infections without symptoms (= healthy colonies)

Summary

5. Prevention: Minimizing transmission, strict control Varroa + other diseases, breeding? Propolis?

4. No patent remedies for viruses, certainly not in the long term control

3. Activation of viruses by Varroa destructor

2. Varroa destructor as a virus vector changes the rules of the game

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Questions

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MLPA Multiplex Ligation Dependent Probe Amplification

• Detection of several viruses in one reaction • LPO = Left Probe Oligo, RPO = Right Probe Oligo • LHS = Left Hybridising Sequence, RHS = Right

Hybridising Sequence

© Lina De Smet

Diagnostics

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How works MLPA?

1. Denaturation: single stranded cDNA 2. Hybridisation

3. Ligation : Ligase-65

4. PCR – single primer pair

LPO RPO

5’ 3’ Target DNA 1

LPO RPO

5’

3’ Target DNA 2

LPO RPO

5’ 3’

LPO RPO

5’ 3’ Target DNA 2 Target DNA 1

Each target : different length

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S1 M S2 S3 S4 S5 M S6 S7 S8 S9 S10 M

M = Marker, S = Sample 140 bp SBV 131 bp SBPV 122 bp BQCV 104 bp ABPV-family 95 bp DWV-family 88 bp CBPV

Diagnostics

Housekee- ping gene