Summary Virology

AN INTRODUCTION TO VIROLOGY
WHAT ARE VIRUSES
Viruses are the smallest, simplest & most abundant form of life on Earth
 Can infect all forms of life (plants, animals, eukaryotes, bacteria)
 Virion: complete, infectious virus particle
 Genetic material: DNA or RNA, ss or ds
o Enclosed in protein coat (= capsid)
 Sometimes lipid membrane (= envelope)
 Viral genome:
o 2 core modules
 Structural proteins  Virion formation (building blocks)
 Non-structural proteins  Genome replication
o Encode for:
 Capsid protein
 Receptor-binding protein
 Viral polymerase
o Doesn’t encode for:
 Protein synthesis machinery
 Proteins for membrane synthesis
 No centromeres or telomeres in standard host chromosomes
 Diameter: 20 – 500 nm
 High abundance, but low relative biomass
 Visualizing: electron microscope

Viruses are obligatory parasites (= can only function after replication in a host cell)
 Depend on host cell for:
 Energy production:
o Mostly ATP, by photosynthesis or metabolism of sugars..
 Protein synthesis
o Ribosomes, transfer RNAs, specific enzymes
o Host ribosomes translate mRNA made by virus
 Reproduction
o Host cell provides building blocks
 Revolution

Viruses are important disease-causing agents
 HIV, Hepatitis, Influenza…
 !!! Not all viruses make us sick
 Most are passengers through our body
 Immune system deals w/ (some) viruses

Viruses have high genome evolution rates
 Evolve rapidly
 Higher mutation rate than eukaryotes & bacteria
 Corona virus has largest RNA genome as we
known for so far

Viruses must be excluded from the tree of life, because:
 Not alive
 No ancestral viral lineages
 No structure derived from a common ancestor




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,ORIGIN OF VIRUSES
Primordial virus world or viral early hypothesis
= viruses are direct descendants of the first replicons that existed
during the pre-cellular stage of the evolution of life
 Viruses existed before cells
 Go very far in evolution, are very ancient

Reductive virus origin
= viruses are the ultimate products of degeneration of ancestral cells
that lost their autonomy & transitioned to obligate intracellular parasitism
 Cells before viruses

Escaped genes hypothesis
= viruses evolved on multiple, independent occasions in different cellular
organisms from host genes that acquired the capacity for autonomous,
selfish replication and infectivity
 Specific genes split off in particles and evolve to viruses

CLASSIFICATION OF VIRUSES: BALTIMORE CLASSIFICATION



Based on the following principles:
 Nature, strandedness, orientation
& topology of nucleic acids
 Nucleotide sequence
 Symmetry of the capsid
 Presence of an envelope
 Virion & capsid dimensions




DNA GENOMES VERSUS RNA GENOMES

DNA genomes RNA genomes
 Genetic material is based on DNA  Genetic material is based on RNA
 Most replicate in the nucleus  A lot of diversity:
 Use host’s DNA- & RNA-synthesizing + RNA-processing  ss or ds
machinery  (+) or (-)
 Dominant in prokaryotes  Linear of segmented
 Classes 1 & 2 of Baltimore system  Most replicate in cytoplasm
 Genome have to encode RNA-dependent RNA
polymerase (RdRp) (cells don’t have this)
 Produces RNA genomes & mRNA from RNA
templates
 Dominant in eukaryotes
 Classes III – VII in Baltimore system




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,CLASSES OF BALTIMORE CLASSIFICATION

Class Features Examples Replication
I  Wide variety in genome size  Adenovirus  Nucleus
(gapped) dsDNA
 Have unfragmented genomes  Herpes virus  DNA  DNA
genomes
 Circular or linear DNA
 No infection of plants
II  Very small & have few genes  Parvovirus  Nucleus
ssDNA genomes
 Replication in nucleus  DNA  DNA
 Circular genomes (dsDNA
 Specific enzyme holding intermediate)
III  Mostly fragmented  Rotavirus A  Cytoplasm
dsRNA genomes
 1 gene encodes for 1  RNA  RNA
protein
 All have icosahedral capsid
symmetry
IV  Most plant & many  Corona virus  Cytoplasm
ss(+)RNA genomes
vertebrate viruses  Yellow fever virus  (+)RNA  (-)RNA
 All linear genomes  Poliovirus
 Small: non-  Hepatitis A virus
enveloped
 Larger: enveloped
 Larger & many plant RNA
viruses have helical capsids
V  Helical nucleocapsids  Ebola virus  Cytoplasm/nucleus
ss(-)RNA genomes
 Some are fragmented  Rabies virus  RNA  RNA
 Not found yet in prokaryotes  Influenza A virus
 Linked to the most  Measles virus
widespread & deadly diseases
VI  Integrated parts of dsDNA in  HIV  Cytoplasm
ss(+)RNA genomes w/
genome of host  RNA  DNA
DNA intermediate
VII  Not originally identified by  Hepatitis B virus  Nucleus &
dsDNA genomes w/ RNA
Baltimore cytoplasm
intermediate
 Gapped dsDNA genomes  DNA  RNA and
 Gaps are filled by then RNA  DNA
reverse transcriptase
& genome is copied
into RNA
intermediate

THE VIROLOGY TOOLBOX
STEP 1: VIRUS ISOLATION & PROPAGATION

 Susceptible cell: functional receptor  may or may not be competent to support viral replication
 Resistant cell: no functional receptor  “
 Permissive cell: capacity to replicate virus  may or may not be susceptible
 Susceptible & permissive cell: can take up & replicate the virus particle for sure

Infection of cells depends on the random collision of virus particles w/ cells
 not all susceptible cells are infected when virus is added

!!! Not all virus particles are infectious
 particle-to-PFU ratio = (# physical viral particles)/(# infectious viral particles)

Multiplicity of infection (MOI): the number of infectious particles added per cell
 MOI = PFU/(number of cells)
 Example: MOI = 10  adding 107 virus particles per 106 cells



STEP 2: VIRUS DETERMINATION
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, PLAQUE ASSAY (QUANTITIVE)
Method:
1. Inoculation of bacteria on nutrient agar in Petri dish
2. Dilutions of a phage suspension
(virus stock, concentration unknown)
3. Add 0,1 ml of dilution to Petri dish
4. Count lysis area = plaque
o Phage binds to bacterial cell
 cell releases progeny phage particles
 taken up by neighboring cells + replicated
 lysis of the cells in a circular area surrounding the original infected cell
 1 plaque = 1 infectious virus particle

HEMAGGLUTINATION (PHYSICAL)
Principle: sialic acid receptors on red blood cells bind to hemagglutinin glycoproteins found on the surface of several viruses
 Advantages:
 Simple
 Inexpensive
 Limitations:
 Controlling incubation times
 RBC type & virus concentration
 Interfering factors in the sample
(pH, T, buffer composition)
 Qualified personal required

ELECTRON MICROSCOPY (PHYSICAL)
 Most recently: cryo-EM

VIRAL ENZYME ACTIVITY (PHYSICAL)
Principle:
 Reverse transcriptase (RT) is used
to synthesize a labeled DNA strand
 DNA labeling can be done w/ radiolabeled
or fluorescently labeled nucleotides
 Labeled DNA can be quantified & the amount
is proportional to the RT activity in the sample

SEROLOGY (PHYSICAL)
= Indirect Immuno Fluorescense Test

 Direct: viral antigen/protein detection
 Indirect: antiviral antibody detection

NUCLEIC ACID DETECTION (PHYSICAL)
= viral genome detection by PCR

VIRUS STRUCTURES
From big to small:
 Virion: infectious virus particle
 Envelope: lipid bilayer derived from host cell (sometimes)
 Nucleocapsid: protein/nucleic acid aggregates within the capsid
 Capsid: protein shell surrounding the genome
 Structural unit: unit from which (nucleo)capsids are build, can contain >1 subunits
 Subunit: single folded polypeptide chain

Functions of structural proteins:
 Protection fragile viral genome
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