Practical Virology Fundamental Laboratory Techniques

The first lab manual for virologists in years, the text includes all basic as well as modern and advanced techniques. 6 distinct methodology sections, ranging from cell to visualization to cloning and sequencing protocols are presented.

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