Practical Virology

Professor C. H. Logue is an experienced arbovirologist and has worked for both the US and UK Governments as well as Academic virology laboratories spanned over three Continents. His research interested include CHIKV, sequencing its full genome for the first time in 2002, CCHFV, having detected the first imported case in the UK, Hantaviruses , having isolated the first strain of SEOV from rats in the UK, and several other virology research interests in South America, Africa and Central Asia. In his role as Virology training lead at Public Health England and Adjunct Professor of Virology at USFQ in Ecuador, he delivers laboratory training in Virology while continuing to teach and contribute to current virology research.

1. An Introduction to the Virology Laboratory

A History of Virology

Virology Laboratory design and equipment used

Definition of Biosafety Levels in Virology Laboratories (BSL1-4)

Virology Laboratory Codes of Practices

Personal Protective Equipment in the Virology Laboratory (BSL1-4)

Storage and transportation of viruses



2. Cell Culture in Virology



Aseptic Technique and working in an MSCII

Preparation of cell culture media

Preparation of continuous cell culture

Preparation of primary cell cultures

Maintenance of cell cultures

Cryopreservation of cells

Quantification of cells

Transformation of bacteria for virus cDNA clone production

Mini and Midi Prep purification from bacterial cultures

Electroporation of cells with virus nucleic acid



3. Cell based virology protocols



Virus propagation

Cell culture

Eggs

Animal models (IN/IC/IP/SC)

Arthropods



Virus Harvest

Cell culture

Animal tissues & blood

Eggs

Environmental samples

Arthropods

Human clinical samples

Arthropod saliva extraction



Plaque Assay ? infectious virus particle titration

Plaque purification

Tissue Culture Infectious Dose (TCID50) - virus titration

Egg Infectious Dose(EID50) - - virus titration

Plaque reduction Neutralisation Test (PRNT) ( see serology-based protocol)

Viral burden by measurement ATP depletion







4. Serology-based virology protocols



Haemagglutination Assay (HA)

Haemagglutination Inhibition Assay (HAI)

Plaque Reduction Neutralisation Test (PRNT)

Immunofluorescence slide preparation (IFA)

IgG /

IgM Indirect ELISA

OgG /

IgM Direct ELISA

Interferon ELISA

Flow cytometry



5. Molecular virology protocols



Nucleic acid extraction (manual / automated / high throughput)

Primer design

Probe design

Thermocycling program design

Polymerase Chain Reaction (PCR)

Reverse Transcription-PCR

Real-Time PCR /

RT-PCR

PCR Platforms (ABI, Roche, BioRad)

Realtime PCR platforms (ABI, Roche, BioRad)

Gel Electrophoresis

Preparation of PCR amplicons for Sanger sequencing

Gel purification of Nucleic acid

Terminal Dye labeling and purification

Preparation of PCR amplicons for NGS

Loop mediated isothermal amplification (LAMP)

Microarray slide preparation



6. Cloning



Virus expression in cDNA clones

Reverse transcription for electroporation

Protein expression in virus vectors

Restriction enzymes



7. Visualization of Virus



Preparation of samples for electron microscopy

Understanding cytopathic effect (CPE) of infected cellc



8. Novel protocols in virology



3D Digital PCR

ViroCyt

Next Generation Sequencing (NGS)

Immunochromatographic assays &Lateral Flow devices

Virus propagation in Plants





9. Annexes

Hazard group and containment list for each virus

Geographical distribution matrix for viruses

Worked examples of calculations for each protocol

List of laboratory equipment and reagents used in each protocol

List of labs with expertise