Standard methods for virus research in Apis mellifera.

Authors: Joachim R de Miranda, Lesley Bailey, Brenda V Ball, Philippe Blanchard, Giles E Budge, Nor Chejanovsky, Yan-Ping Chen, Laurent Gauthier, Elke Genersch, Dirk C de Graaf, Magali Ribière, Eugene Ryabov, Lina De Smet, Jozef J M van der Steen.

Table of contents

 

Authors
Summary
1. Introduction
   1.1. Honey bee viruses

      1.1.1. Acute bee paralysis virus /Kashmir bee virus /Israeli acute paralysis virus
      1.1.1. Black queen cell virus
      1.1.3. Aphid lethal paralysis virus & Big Sioux River virus
      1.1.4. Deformed wing virus /kakugo virus /Varroa destructor virus-1 /Egypt bee virus
      1.1.5. Sacbrood virus /Thai sacbrood virus
      1.1.6. Slow bee paralysis virus
      1.1.7. Chronic bee paralysis virus /satellite virus
      1.1.8. Cloudy wing virus
      1.1.9. Bee virus X /Bee virus Y
      1.1.10. Lake Sinai virus-1 /Lake Sinai virus-2
      1.1.11. Arkansas bee virus & Berkeley bee virus
      1.1.12. Apis mellifera filamentous virus
      1.1.13. Apis iridescent virus
   1.2. Definitions: pathogenicity vs virulence; incidence vs prevalence
   1.3. Virus replication and variation

      1.3.1. The mathematics of virus replication and transmission
      1.3.2. Virus variability and evolution
2. Virus surveys
   2.1. Introduction
   2.2. Types of survey

      2.2.1. Passive surveillance
      2.2.2. Active surveillance
3. Statistical aspects
   3.1. Introduction
   3.2. Statistical distribution of virus data

      3.2.1. Log-transformation
      3.2.2. Zero values
4. Virus sample management
   4.1. Introduction
   4.2. What, where and when to sample

      4.2.1. What to sample?
      4.2.2. Where to sample?
      4.2.3. When to sample?
   4.3. Sample collection
      4.3.1. Adult bees
      4.3.2. Pupae
      4.3.3. Larvae
      4.3.4. Eggs
      4.3.5. Extracted guts
      4.3.6. Drone endophallus and semen
      4.3.7. Faeces
      4.3.8. Dead colonies
   4.4. Sample transport
      4.4.1. Freezing
      4.4.2. Ice
      4.4.3. Live transport
      4.4.4. Chemical stabilizers
      4.4.5. Sample collection cards
   4.5. Long-term sample storage
5. Virus propagation
   5.1. Introduction
   5.2. Starting material
   5.3. Oral propagation

      5.3.1. Larvae
      5.3.2. Adults
   5.4. Injection propagation
      5.4.1. Pupae
      5.4.2. Adults
   5.5. Tissue culture
      5.5.1. Virus infection
   5.6. Full-length infectious virus clones
      5.6.1. Full-length viral RNA synthesis strategies
      5.6.2. Protocol
         5.6.2.1. Full-length reverse transcription
         5.6.2.2. Amplifying full-length viral RNAs
         5.6.2.3. Cloning full-length viral RNAs
         5.6.2.4. Confirmation of full-length clones
         5.6.2.5. Synthesizing full-length viral RNA
         5.6.2.6. Confirmation of Full-length Viral RNA
6. Virus infectivity assays
7. Virus purification
   7.1. Introduction
   7.2. Protocols

      7.2.1. Primary extract
      7.2.2. High speed centrifugation
      7.2.3. Gradient Centrifugation
         7.2.3.1. Sucrose gradients
         7.2.3.2. Caesium chloride gradients
         7.2.3.3. Fractionation
8. Virus sample processing
   8.1 Introduction
   8.2. Sample homogenisation

      8.2.1. Bead-mill homogenizers
      8.2.2. Blender
      8.2.3. Mortar and pestle
      8.2.4. Mesh bags
      8.2.5. Micropestle
      8.2.6. Robotic extraction
   8.3. Nucleic acid extraction
      8.3.1. Protocol 1 – affinity column purification
      8.3.2. Protocol 2 – TRIzol extraction and isopropanol precipitation
   8.4. Nucleic acid quality assessment
9. Virus detection
   9.1. Introduction
   9.2. Enzyme-Linked ImmunoSorbent Assay (ELISA)

      9.2.1. Normal ELISA
      9.2.2. Sandwich ELISA
      9.2.3. Protocols
         9.2.3.1. Sample preparation
         9.2.3.2. ELISA
         9.2.3.3. Sandwich ELISA
         9.2.3.4. Development
         9.2.3.5. Controls
   9.3. RT-(q)PCR
      9.3.1. Primer design
      9.3.2. Detection and analysis of PCR products
         9.3.2.1. “End-point” vs “real-time” detection
         9.3.2.2. Cycles and thresholds
         9.3.2.3. Detection chemistry
      9.3.3. Assay optimization
         9.3.3.1. Reverse transcription
         9.3.3.2. One-Step/Two-Step RT-PCR
      9.3.4. Protocols
         9.3.4.1. Reverse transcription
         9.3.4.2. Two-Step RT-qPCR
      9.3.5. Quantitation controls
         9.3.5.1. External reference standards
         9.3.5.2. Internal reference standards
      9.3.6. Multiple assays
      9.3.7. Multiplex RT-(q)PCR
   9.4. Microarrays
10. Virus replication
   10.1. Introduction
   10.2. Strand-specific RT-qPCR     

      10.2.1. Thermostable reverse transcriptases
      10.2.2. Chemical blocking of RNA 3’ ends
      10.2.3. Tagged cDNA primers
      10.2.4. Removal of tagged-cDNA primers from the cDNA reaction
      10.2.5. Inactivating tagged-cDNA primers in the cDNA reaction
      10.2.6. Dilution
      10.2.7. Strand-specific real-time RT-qPCR
      10.2.8. Protocols
         10.2.8.1. High temperature reverse transcription
         10.2.8.2. Exonuclease-I digestion of tagged primer
         10.2.8.3. Column purification of cDNA
         10.2.8.4. OneStep PCR
         10.2.8.5. Real-time qPCR
      10.2.9. Controls
   10.3. Multiplex Ligation-dependent Probe Amplification (MLPA)
      10.3.1. Introduction
      10.3.2. Protocol
         10.3.2.1. Primer and probe mixtures
         10.3.2.2. Reverse transcription
         10.3.2.3. Hybridisation of MLPA half-probes
         10.3.2.4. Ligation of MLPA half-probes
         10.3.2.5. PCR amplification of MLPA probes
      10.3.3. Fragment analysis
      10.3.4. Controls
11. Virus variation
   11.1. Introduction
   11.2. Protocols

      11.2.1. Nuclease protection assays (RPAs and SNPAs)
      11.2.2. Gel retardation assays (SSCP and DGGE)
      11.2.3. High Resolution Melting (HRM) analysis
      11.2.4. Sequencing
12. Quality control
   12.1 Introduction
   12.2. Assay selection and validation

      12.2.1. Analytical specificity
         12.2.1.1. Analysis in silico
         12.2.1.2. Experimental specificity
      12.2.2. PCR detection limit
      12.2.3. qPCR dynamic range and quantitation limit
   12.3. Method validation
      12.3.1. Method detection limit
      12.3.2. Method diagnostic sensitivity and diagnostic specificity
      12.3.3. Method quantitation limit and accuracy profile
   12.4. Laboratory Validation
      12.4.1. Training and accreditation
      12.4.2. Inter-laboratory proficiency testing
13. Future perspectives
14. Acknowledgements
15. References
Table 1
Table 2
Table 3
Table 4
Table 5
Table 6
Table 7