Lecture 2 - Viral Agents 1: Nature and Classification of Viruses Flashcards
What is a virus?
An obligate intracellular molecular parasite
Eukaryotic organelle that poliovirus is a similar size to
Ribosome (20nm). Polio virion is 30nm
Range of virion sizes
20nm to 300nm
Negative staining stains
Potassium phosphotungstate
Ammonium molybdate
Qualities of good negative stains for viral EM
1)
2)
1) Highly soluble (so don’t crystallise as they dry)
2) Electron-dense
Cryo-electron microscopy advantage
Transmission EM without electron-dense negative stain.
Therefore no distortion from sample prep
Cryo-electron microscopy
1)
2)
3)
1) Prepare virus by freezing in vitreous (uncrystallised) water
2) Electron microscopy at -160C
3) Two-dimensional projections (Fourier transformations) assembled into a three-dimensional image (inverse Fourier transform)
What is cryo-electron microscopy useful for?
Imaging viruses with a labile envelope
What are X-ray crystallography images constructed from?
Electron-density maps
Protein subunits of capsid
Capsomeres
Nucleocapsid
Proteins most closely associated with nucleic acid
Matrix
Some viruses have a protein layer beneath the envelope that connects the capsid and envelope glycoproteins
Capsid shapes
1)
2)
3)
1) Helical symmetry
2) Icosahedral
3) Complex symmetry
Icosahedral symmetry
20 faces
Three axes of symmetry - 2-fold, 3-fold, 5-fold
Examples of viruses with icosahedral symmetry
1)
2)
3)
1) Adenovirus
2) Herpesvirus
3) Pappilomavirus
Advantage of a lipid envelope
Can leave host cell without killing it
What are viruses divided into?
Family, genera, species
How are viral families distinguished?
Kind of genome, genome arrangement, replication strategy, virion morphology (capsid symmetry, virion size, envelope)
What do viral family names end in?
-viridae
What ends in -viridae?
Viral family names
How are viral genera distinguished?
Size of genome, genome sequence differences, number, size of proteins, serological reactivity, host range and disease
What do viral genus names end in?
-virus
What ends in -virus?
Names of viral genera
How can DNA and RNA viruses be distinguished?
1)
2)
3)
1) Infect cells in presence of 14C-thymidine and 3H-uracil.
2) Purify viral particles produced in cells
3) Use a scintillation counter (radiation detector) to determine whether a virus contains 14C-thymidine (DNA) or 3H-uracil (RNA)
How to determine whether a virus contains ssRNA or dsRNA 1) 2) 3) 4) 5)
1) Label viral RNA during growth
2) Extract viral RNA from purified particles
3) Divide into two portions. Add RNase A/T1.
4) RNase A/T1 only digests ssRNA
5) Trichloroacetic acid (TCA) is used to precipitate RNA. Only RNA polymers will precipitate (if ssRNA has been digested, won’t precipitate)
Way to solubilise proteins and lipids in viral purification
Sodium dodecyl sulphate plus or minus proteinase K
Viral purification method
1)
2)
1) Solubilise proteins and lipids (sodium dodecyl sulphate/proteinase K)
2) Phenol extraction
Molecule separations in phenol extraction
1)
2)
1) Aqueous phase - Nucleic acids
2) Phenol phase - Proteins
Do all viruses cause disease?
No. Most infections are subclinical
Example of DNA helical-symmetrical virus
None. No DNA helical-symmetrical viruses exist
Modified Koch's postulates for viruses 1) 2) 3) 4)
1) Virus should have regular association with disease in question
2) Virus must be characterised (isolated from cell culture, genetically or immunologically distinguished from other viruses)
3) Clinical syndrome should be reproducible in animals or human volunteers
4) Virus should be reisolated from test subject
Examples of enteric viruses
Rotavirus
Calicivirus
Some adenoviruses
Examples of respiratory viruses
Orthomyxovirus
Rhinovirus
Some paramyxoviruses, coronaviruses, adenoviruses
Arboviruses
Infect insects, which then infect humans
Viruses which infect insects and then humans
Arboviruses
Hepatitis viruses that are spread enterically
A and E
Hepatitis viruses spread sexually or through blood
B, C, D
