HOST CELL SURFACE RECEPTOR MOLECULE(S) FOR DEFORMED WING VIRUS (DWV) THAT

MEDIATE HOST-PATHOGEN INTERACTIONS IN HONEYBEES

By

FAITH AKINYI OBANGE

INTRODUCTION• Apiculture industry generates employment by producing honey and beeswax for commercial

purposes (FAO, 2006)

• Honey bees (Apis mellifera) are important for crop pollination and maintaining biodiversity of trees through cross pollination of tree flowers (FAO, 2008)

• ‘One of every three bites of food eaten worldwide depends on pollinators, especially bees, for a successful harvest’(Grossman, 2013)

• The global economic value of the pollination services provided by honeybees is estimated at €22.8 - 57 billion (UNEP, 2010)

• Viral infections, most notably Deformed Wing Virus (DWV) Syndrome, present a significant threat to bee health

• There is need for a better understanding of DWV in order to develop effective control strategies against it(Yan et al., 2007)

EPIDEMIOLOGY OF DWV SYNDROME• DWV syndrome is the most widespread honeybee viral disease in Europe and it has recently

been found in the USA (Y P Chen, Higgins, & Feldlaufer, 2005).

• DWV was first reported in Algeria and North Africa in 2013 (Loucif-Ayad et al.,2013). • DWV syndrome is closely associated with the ectoparasitic Varroa destructor mite (Kuster,

Boncristiani, & Rueppell, 2014)

• DWV is linked to colony collapse disorder (CCD) (Navajas et al., 2008that is most notably characterized by complete absence of adult bees (Council, 2007).

• The U.S lost 59% of its honeybee colonies colonies between 1947 and 2005 while Europe lost 25% of its colonies between 1985 and 2005

• Over the years, there has been an increase in the proportion of pollination dependent agricultural crops (Sammataro et al., 2000)

• This raises serious concerns about the declining availability of the pollination services provided by honeybees

ETIOLOGY OF DWV SYNDROME• DWV syndrome is caused by the DWV which is an RNA virus of the genus Iflavirus (Y P Chen

et al., 2005)

• Its genomic RNA is 10,140 nucleotides long containing a single large ORF encoding a 328-kDa polyprotein comprising three major structural proteins: VP1 VP2 and VP3 (Lanzi et al., 2006).

• DWV was first detected in the honey bee by RT-PCT in Moscow, Russia (Udina et al., 2010)• Horizontal transmission of overt DWV:

i. infection of adult bees through DWV- carrying Varroa destructor mitesii. infection of nurse bees through their cannibalizing DWV-infected pupae (Möckel et al.,

2011) iii. larvae feeding on DWV contaminated larval food (Yue & Genersch, 2005)

• Vertical transmission of covert DWV:i. infection from queens to offspring (Yue et al., 2007)ii. infection from drones to offspring (Y P Chen et al., 2006)

• It is unknown whether venereal transmission of DWV occurs during natural mating of queen and drone honeybees (J R de Miranda et al., 2008)

SIGNS AND SYMPTOMS OF DWV SYNDROME

• DWV syndrome clinical symptoms most notably include (Joachim R de Miranda et al., 2010) :i. pupal death ii. emergence of non-viable, short-lived adults with :a) deformed wingsb) shortened abdomen c) discolouration

• The manifestation of symptoms depends on (Schöning et al., 2012):i. DWV transmission by Varroa destructorii. Replication of DWV in Varroa destructor iii. The DWV titre in the Varroa destructor

• Varroa infested bees often have a short, fat body that (FAO, 2006):i. hampers the functioning of their glandsii. increases their susceptibility to pesticides iii. may considerably reduce the semen production of drones

ROLE OF Varroa destructor MITES IN DWV TRANSMISSION

• Varroa destructor infestation immunosuppresses honey bees and leads to the activation of overt/ latent DWV replication (Shen et al., 2005)

• Varroa destructor mite’s biting activity in itself contributes to the increase in DWV titers (Kuster et al., 2014) through its salivary secretions(FAO, 2006)

• This is by suppressing expression of the honeybee immunity-related genes (Xiaolong et al., 2005)

• Exposure to bacteria further lowers survivorship of mite-parasitized worker bees since the Varroa destructor mite interferes its immunocompetence (X. Yang et al., 2006)

• V. destructor acts as a mechanical and biological vector of DWV enabling the replication of DVW in mites prior to its vectoring into honey bee (Genersch et al., 2010)

• Both adult and immature mite progeny acquire DWV from DWV-infected host bees; not from their mother mites (Nordström, 2003)

TREATMENT OF DWV SYNDROME• No vaccine or medication exists for DWV syndrome

• DWV’s most effective management remains elimination of the Varroa destructor mite though an integrated pest management (IPM) plan (Highfield et al., 2009)

RESISTANCE TO Varroa destructor MITE AND DWV

• A population of pure breeding Chinese black honeybees possess some natural resistance to Varroa destructor mite indicating the importance for the conservation of the black honeybees in China (Peng et al., 2015)

• DWV has been detected in and poses a serious threat to bumble bee populations (Genersch et al., 2006); threatening ecological biodiversity

• Africanized honey bees have a resistance to Varroa destructor mite (Harbo et al., 2001)

• This is due to the grooming behavior of the bees keeping mite infestation low in the colonies (Calderón et al., 2010)

Varroa destructor HOST-PARASITE INTERACTIONS

• Host-parasite interactions involve (Locke, 2012) :i. host tolerance which is the host’s ability to reduce the effect of the parasite ii. host resistance which is the host’s ability to reduce the fitness of the parasite

• Chemical controls applied to manage Varroa destructor mites remove selective pressures

necessary for host/parasite adaptations towards a stable host-parasite relationship in which host bees do not need mite control treatment (Locke, 2012)

• Such a stable host-parasite relationship was found in South Africa (Fries, 1997)

DWV HOST-PATHOGEN INTERACTIONS• Host-pathogen interactions can take the form of (Ryabov et al., 2014):

i. asymptomatic infections characterized by restricted pathogen replication, consequent diminished odds of host-to host horizontal transmission and prolonged survival of the infected host which increases the odds of DWV vertical transmission

ii. symptomatic infections characterized by rapid pathogen replication, increased virulence and maximized host-to-host horizontal transmission

• The interaction between DWV and the Varroa mite turns a docile virus into a rapidly replicating killer virus through a strong down-regulation of the vital transcription factor NF-kB (Nazzi et al., 2012)

• Literature on the host cell surface receptor molecules utilized by DWV to mediate its host-pathogen interactions in honeybees is scarce

• This study sets forth with the goal of identifying the host cell surface receptor molecule(s) for Deformed Wing Virus (DWV) that mediates its host-pathogen interactions in honeybees

PROBLEM STATEMENT

• DWV syndrome is a deadly disease threatening the global honeybee population, crop pollination, plant biodiversity, the apiculture industry and global food security.

• Studies have been conducted on its epidemiology and etiology.

• However, there exists a knowledge gap concerning the host cell surface receptor molecules for DWV that mediate its host-pathogen interactions in honeybees.

JUSTIFICATION • DWV infection presents a direct threat to bee health, the apiculture industry, global food security and

biodiversity

• Thus, there is need for a better understanding of DWV virus host-pathogen interactions in honeybees.

• In order to accomplish this, there is need for application of molecular techniques for the accurate cloning, expression and production of antibodies against the recombinant major structural proteins of DWV as well as identification of host cell surface receptor molecule(s) for DWV in honeybees.

• Accurate identification of the receptor molecule requires a method that allows the receptor protein to retain its native state and enables one to study how post-translational modifications affect protein-protein interactions, examine interaction sequences using synthetic peptides as probes and identify protein-protein interactions without the need for antigen-specific antibodies.

• Thus, the most appropriate techniques to apply would be Far Western Blot assay and Virus Overlay Recombinant Protein Protein-Binding Assay (VOPBA).

• The knowledge generated could be used to design novel control strategies for DWV

RESEARCH QUESTIONS1.Is it possible to clone and express the recombinant major structural proteins (VP1 and VP2) of DWV?2.Which antibodies are produced against the expressed recombinant major structural proteins (VP1 and VP2) of DWV?3.What is the host cell surface receptor molecule(s) for DWV in honeybees?

HYPOTHESIS DWV does not bind to any cell surface receptor molecule(s) in the honeybee.

OBJECTIVES Broad objectiveTo perform the molecular characterisation of host cell surface receptor molecule(s) for Deformed Wing Virus (DWV) that mediate host-pathogen interactions in honeybees. Specific objectivesTo clone and express the recombinant major structural proteins (VP1 and VP2) of DWVTo prepare antibodies against the expressed recombinant major structural proteins of DWV To identify host cell receptor molecules for DWV in honeybees

EXPECTED OUTPUTThe knowledge generated could be used to design novel control measures for bee populations at risk to DWV virus infections.

MATERIALS AND METHODSSTUDY DESIGN • Prospective study that will be performed by testing haemolymph and GIT homogenate

samples collected from a worker honeybee presenting clinical signs of DWV syndrome.

• Study will be limited to identification of the host cell surface receptor molecules utilized by DWV to mediate its host-pathogen interactions in honeybees only.

• Identification of the host cell surface receptor molecules utilized by DWV to mediate its host-pathogen interactions in Varroa destructor mite will be delimited from this study.

SAMPLING• Sampling will be performed randomly to generate a single sample identification number

using R statistical software as applied by Ross Ihaka (Ihaka & Gentleman, 1996).

MATERIALS AND METHODS Cont…1. TO CLONE AND EXPRESS THE RECOMBINANT MAJOR STRUCTURAL PROTEINS (VP1

AND VP2) OF DWV • Viral RNA Isolation : ISOLATE II RNA Mini Kit (Bioline) • cDNA synthesis: Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT)

(Promega) used with random primers and oligo (dT) primers• DNA amplification: High-fidelity PCR via VELOCITY DNA Polymerase (Bioline), with

target DNA sequences will being DWV P1, P2 and GFP reporter gene • Gel electrophoresis: Agarose gel electrophoresis• PCR amplicon isolation: ISOLATE II PCR clean up & Gel extraction Kit (Bioline)• Cloning: PCR amplicons will be cloned into pGEM-T Easy Vector System (Promega) • Sub-cloning: Insert subcloned into the destination vector pIZ/V5-His (Invitrogen).• Sequencing :To confirm the integrity of the cloned ORF (Illumina Miseq platform).• Protein expression: pIZ/V5 subjected to cationic lipid mediated transfection into

Sf21 cell and subsequent transient expression of the cloned insert’s protein• Protein purification: HIS-Select® Spin Columns• Protein detection: SDS-PAGE, Proteins transferred onto nitrocellulose membranes,

membranes probing with affinity-purified Anti-6xHis IgG monoclonal Antibody, incubation with a secondary antibody conjugated to horseradish peroxidase, Signal visualisation with an enhanced chemiluminescence detection system

MATERIALS AND METHODS Cont…2. TO PREPARE ANTIBODIES AGAINST THE EXPRESSED RECOMBINANT MAJOR STRUCTURAL PROTEINS OF DWV i. Production of mouse IgG polyclonal antibodies • Immunogen prepared via EDC Conjugation (carboxyl and amine crosslinking) of the

expressed cloned recombinant DWV structural proteins to the carrier protein PEGylated mcKLH (Thermo®) with Alum used as an adjuvant

• Pre-immune serum from the mouse will be collected and freeze stored for later use as a blank when performing ELISA screening after immunization.

• 50 to 100μg of immunogen (equal to 100 to 200μL of antigen-adjuvant mixture) will be injected intraperitonealy into each mouse.

• 14 days after immunization, the mice will be given a booster injection of the same volume and composition.

• 21 days after immunization, the mice will be test bled under anaesthesia through the tail vein and ELISA performed to assay antibody response.

• The mice will receive a booster injection on the 28th day if necessary. • This schedule of alternating schedule of booster injections and test bleeds will continue

until a satisfactory response is observed.

MATERIALS AND METHODS Cont…ii. Purification of IgG polyclonal antibodies • Produced IgG proteins will be purified using Melon™ Gel Purification Products (Thermo®)

iii. Antibody detection• SDS-PAGE, Proteins transferred onto nitrocellulose membranes, membranes probing with

affinity-purified Anti-6xHis IgG monoclonal Antibody, incubation with a secondary antibody conjugated to horseradish peroxidase, Signal visualisation with an enhanced chemiluminescence detection system

MATERIALS AND METHODS Cont…3. TO IDENTIFY HOST CELL RECEPTOR MOLECULES FOR DWV IN HONEYBEESi. Cell membrane receptor protein isolation• Isolation of cell membrane proteins from Honeybee haemolymph and GIT homogenate via the

ProteoExtract™ Native Membrane Protein Extraction Kit (Merck Millipore)ii. Detection of Receptor-ligand interactions

a) Virus Overlay Protein-Binding Assay (VOPBA)• To identify cell polypeptides involved in virus binding, a VOPBA assay will be carried out.• Isolated membrane proteins will be subjected to native PAGE and transferred onto nitrocellulose

membranes using a semi-dry blotting apparatus in transfer buffer (48 mM Tris, 29 mM glycine and 20% methanol).

• After overnight renaturation of transferred proteins with 4% BSA in PBS at 4 °C, membranes will be blocked for 1h at room temperature with 5% skimmed milk (Gibco-BRL) in PBS and re-washed three times with PBS.

• Membranes will be incubated overnight with tagged DWV in MEM supplemented with 10% FBS at room temperature with gentle rocking.

• After washing four times for 15 min with 2% BSA in PBS and once with 1% Nonidet P-40 in PBS at room temperature, membranes will be dried and autoradiographed.

• Total cell membrane proteins separated by 20% native PAGE, purified proteins separately electroeluted from gel and candidate receptor protein(s) identified by MS.

• Results will be compared to those obtained via Far Western Blot assay.•

MATERIALS AND METHODS Cont…b) Far Western Blot assay • To avoid false positive and false negative protein-protein interraction, gel

electrophoresis will be performed under native conditions (i.e., non-denaturing and without reducing agent).

• Proteins will be transferred from the gel to a nitrocelluse membrane.• A blocking buffer will be added to reduce nonspecific binding and aid in protein

renaturation. Membranes will be blocked with 5% dry milk in Tris-buffered saline containing Tween 20 for 30 min at room temperature (22ºC).

• Membranes will be probed with affinity-purified Anti-candidate_receptor_protein monoclonal Antibody (dilution 1: 5000 in TBS-T) for 3 h at room temperature (22ºC). For controls, identified_receptor antibodies will be pre-absorbed to the recombinant receptor fusion protein before immunostaining.

• Membranes will be washed with TBS-T, followed by incubation with a secondary antibody conjugated to horseradish peroxidase (1: 10 000). Signals will be visualized with an enhanced chemiluminescence detection system.

WORK PLANMONTH

TASK

OCT NOV DEC JAN FEB MAR

Project proposal development

Sampling

Cloning of the recombinant major structural proteins of DWV

Expression of the recombinant major structural proteins of DWV

Antibody preparation against the expressed recombinant major structural proteins of DWV

Identification of host cell receptor molecules for DWV in honeybees

Statistical data analysis

Thesis preparation and submission

BUDGETPack size Quantity Unit price Cost (KSh.)

cDNA synthesis and DNA amplification

ISOLATE II RNA mini kit (Bioline) 100 1 64,480.00 64,800.00

PCR primers - 6 2,600.00 156,000.00

Ultrapure™ DNAse/RNAse-Free Distilled Water

10x500ml 2 13,197.60 26,395.20

Consumables  - - - 195,000.00

Cloning of the recombinant major structural proteins of DWV

- - - 200,000.00

Antibody preparation against the expressed recombinant major structural proteins of DWV

- - - 50,000.00

Identification of host cell receptor molecules for DWV in honeybees

- - - 10,000.00

Sequencing  -  - 1,060  890,000 .00

Thesis preparation and printing - - - 10,000.00

Transport  - - - 50,000.00

GRAND TOTAL

 

1,452,195.00