INTRODUCTION
CLASSIFICATION OF VIRUSES
The ICTV classification: groups viruses by characteristics
• Genome: RNA or DNA, single or double stranded, orientation (+ or -) and topology (linear, gapped or circular)
• Nucleotide sequence! --> if viruses have the same sequence, they must be related
• Symmetry of the capsid
• Presence or absence of a lipid membrane (envelope)
• Dimensions of the virion and the capsid
Virus taxonomy: phyla, subphyla, classes, orders, suborders, families, subfamilies, genera, subgenera and species
! ICTV uses taxonomic units: order (-virales), family (-viridae), genus (-virus) and species (-virus)
--> not all viruses are assigned to an order (big group with common characteristics)
well-defined so you can talk about the same virus worldwide BUT it changes quite often
The Baltimore system
Viral genomes (all viruses) must make mRNA that can be read by host ribosomes.
All viruses on the planet follow this rule, no known exception.
BUT the way how they make mRNA is quite different --> basis for the Baltimore classification
Baltimore groups viruses based on how they make mRNA from the genome (NOT on which viral genome there is)
(7 classes of viruses that have a different type of genome and a different way of making mRNA)
By just knowing the genome structure, the Baltimore classification system enables to deduce
how mRNA is made from the viral genome and how the genome is copied to make more genomes for packaging in virions.
Definitions/rules underlying the Baltimore system:
• mRNA (‘ribosome ready’) is always the plus (+) strand
• DNA of equivalent polarity is also the (+) strand
• RNA and DNA complements of (+) strands are negative (-) strands
• Not all (+) RNA is mRNA! (e.g. HIV)
Viruses with DNA genomes (classes I and II)
• The host genetic system is based on DNA --> many DNA viruses compete with the host for host processes
• However, almost all viral DNA genomes are NOT like cell chromosomes
• In contrast to RNA viruses, most DNA viruses replicate their genomes in the nucleus (pox viruses replicate in cytoplasm)
and utilize the host DNA- & RNA-synthesizing machinery, along with the host’s RNA-processing machinery
Because DNA viruses utilize enzymes present in the nucleus of infected cells to make new DNA and mRNA,
almost all DNA viruses will travel to the nucleus upon infection where their replication will happen.
• They dominate in the prokaryotic world (most bacteria are infected by DNA viruses)
Viruses with RNA genomes (classes III – VII)
• Unique because their genetic information is encoded in RNA
• Diverse: ss or ds, (+) or (-) sense, linear or segmented (= different genomic strands next to each other)
• Eukaryotic cells have no RNA-dependent RNA polymerase (RdRp) --> RNA virus genomes have to encode RdRp
--> almost all RNA viruses will replicate in the cytoplasm because they don’t need enzymes from the host’s nucleus
(a lot of antiviral innate immune proteins are all active against RNA viruses that replicate in the cytoplasm)
• RdRp produces both new RNA genomes as well as mRNA from RNA templates (the genome)
• They dominate in the eukaryotic world
What information is encoded by the viral genome?
Minimally: a gene encoding the capsid protein
a gene encoding a receptor-binding protein (for non-enveloped viruses: the capsid protein is the protein that binds to the receptor)
a gene encoding for a viral polymerase (it does not always need this enzyme because sometimes it uses the cellular polymerase)
Most of them encode for gene products and regulatory signals for: protein synthesis (to block cellular protein synthesis),
replication of the viral genome, assembly and packaging of the genome, regulation and timing of the replication cycle
(viruses enter and will first make proteins from the genome and afterwards copy the genome to make more genomes),
modulation of host defenses (stopping the immune system just long enough for the virus to replicate), spreading to other
cells and hosts, …
What information is NOT encoded by the viral genome? (not in viruses infecting eukaryotic cells)
- no genes encoding the complete protein synthesis machinery (eIFs,tRNAs)
- no genes encoding proteins involved in membrane biosynthesis
- no classical centromeres or telomeres found in standard host chromosomes
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,BALTIMORE CLASSIFICATION
The Baltimore system groups viruses into 7 distinct classes based on the type of nucleic acid in the virus (DNA or RNA), the strandedness
of the nucleic acid (single-stranded or double-stranded), and the method the virus uses to produce mRNA for protein synthesis.
1. Class I = dsDNA viruses
- replication in nucleus (exception: Poxviruses)
--> the viral DNA is directly transcribed into mRNA using the cellular or viral RNA-dependent DNA polymerase
- examples: Polyomaviruses, Papillomaviruses (cellular RdDp)
Herpesviruses, Adenoviruses, Poxviruses (viral RdDp) --> replicate in cytoplasm since they don’t need cellular enzymes
! all dsDNA viruses have unfragmented genomes --> all have a genome of one strand (circular or linear)
2. Class II = ssDNA viruses
- replication in nucleus
--> ssDNA in converted into dsDNA by cellular DNA-dependent DNA polymerase, which is then transcribed into mRNA
- examples: Parvoviruses (other ssDNA viruses do not infect humans)
! DdDp is not present in resting cells: ssDNA viruses usually infect rapidly dividing cells (epithelial cells of B-cells) to replicate
! most ssDNA viruses have very small circular genomes and no envelope
3. Class III = dsRNA viruses
- replication in cytoplasm
--> dsRNA is transcribed into mRNA by the viral RNA-dependent RNA polymerase (encoded by the RNA genome)
- examples: Reoviruses (including Rotaviruses), Birnaviruses
! dsRNA = one of the strongest triggers of innate immune response and inflammation
--> there aren’t many dsRNA viruses because our innate immune system has evolved to react very violently against the presence of dsRNA
--> these viruses will not fully release their genome when they enter the cell (it always remains covered by some part of the nucleocapsid)
! most dsRNA viruses have fragmented genomes / all dsRNA have icosahedral capsids
4. Class IV = (+) ssRNA viruses
- replication in cytoplasm
--> the viral RNA is transcribed into (–) strand RNA, which is then transcribed into mRNA by vRdRp
- examples: Astroviruses, Caliciviruses, Coronaviruses (like SARS-CoV-2), Flaviviruses (like West Nile virus),
Picornaviruses (like poliovirus), Arteviruses, Togaviruses
! The viral RNA serves directly as mRNA, which is translated into viral proteins by the host cell’s ribosomes
BUT Baltimore does not say anything about whether the genome can be used as mRNA to make a protein
--> Baltimore is based on how the virus makes new mRNA from the genome
! all (+) ssRNA viruses have linear genomes / dsRNA viruses with small genomes have no envelope vs. viruses with larger RNA genomes are enveloped
5. Class V = (–) ssRNA viruses
- replication in cytoplasm (exception: some viruses, like Influenza, will replicate in the nucleus)
--> the viral RNA is transcribed into (+) mRNA by the viral RNA-dependent RNA polymerase
- examples: Arenaviruses, Orthomyxoviruses (like influenza), Paramyxoviruses,
Bunyaviruses, Filoviruses, Rhabdoviruses (like rabies)
! some (–) ssRNA viruses have fragmented genomes / all (–) ssRNA viruses have helical nucleocapsids / some genera have ambisense RNA genomes
6. Class VI = (+) ssRNA viruses with a DNA intermediate
- replication in cytoplasm
--> the viral RNA is reverse transcribed into dsDNA by viral reverse transcriptase (via DNA-RNA-intermediate),
which is then incorporated into the host genome and transcribed into mRNA
- examples: Retroviruses (like HIV)
! the positive RNA genome is not directly recognized as mRNA by ribosomes in the cytoplasm
7. Class VII = dsDNA viruses with a DNA intermediate
- replication in cytoplasm
--> the viral DNA is transcribed into (+) ssRNA, which then reverse transcribed back into DNA by viral reverse transcriptase,
the dsDNA is then transcribed into mRNA
- examples: Hepadnaviruses (like Hepatitis B virus), Caulimoviruses
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,THE VIROLOGY TOOLBOX
Virus diagnosis methods: is the virus present or not? (two main methods to study viruses)
1. Direct methods: detect the virus or a component of the virus itself
- virus isolation: take a sample from the patient and bring it on susceptible cells (you know which cell line allows to grow this virus)
--> look at infection: if the virus grows, you know the virus is there
(usually combined with another method to confirm that it is this virus because even a specific cell line will grow several types of viruses)
- viral genome detection (PCR)
- antigen detection
2. Indirect methods: detect immune response against the virus: if there are Ab against the virus in bodily fluids,
you have been exposed to the virus (you don’t know if you have the virus at that time, maybe it was 10 years ago)
Virus isolation and propagation
• Before cell culture: dependent on laboratory animals (that are susceptible)
--> first study on viruses where done in chickens (rabbits and chickens have a rapid generation time)
• Before cell culture and after laboratory animals: in embryonated chicken eggs
--> inoculating the virus at specific sites in the egg allowed replication of the virus
! some viruses grow very badly on cell cultures so this method is still used:
- influenza vaccines are grown in this way because there is no method with cell lines to efficiently grow the virus
- if a new virus is discovered, it is grown in chicken eggs because it is not yet known whether the virus grows on cell lines
• In cell culture: adding viruses to cell lines induces CPE = cytopathic effect (you don’t see the virus but the result of the infection)
--> rounding of cells, shrinkage, fusion and syncytia formation, aggregation of cells, loss of adherence, cell lysis/death
(when a virus infects a cell line, the effect of virus replication on this specific cell line can be seen)
! Not all viruses cause CPE, but inclusion bodies can be seen as IC granules that are sites of replication or assembly
! primary cell lines --> problem of contact inhibition: you cannot keep on replication the viruses, they stop growing, so in the lab immortalized cell lines are used
Some important definitions and concepts
• A susceptible cell has a functional receptor for a given virus - the cell may or may not be able to support viral replication
(virus needs to be able to bind to a receptor on the cell surface --> if receptor is present, cell is susceptible but does not mean full replication cycle can happen)
• A resistant cell has no receptor - it may or may not be competent to support viral replication
• A permissive cell has the capacity to replicate virus - it may or may not be susceptible (may or may not have the receptor)
! all the enzymes and machinery need to be present inside the cell to sustain the replication of a specific virus
For example, ssDNA viruses need cells that express the DNA dependent DNA polymerase to make dsDNA from ssDNA
--> if a ssDNA virus infect a cell with a functional receptor but that cell is not a rapidly dividing cell (the cell is not permissive)
it will not allow the full replication cycle to happen, so the virus will bind to the cell, will be internalized and the genome
will be released BUT then it stops (there is no infection)
• A susceptible AND permissive cell is the only cell that can take up a virus particle and replicate it
! both susceptible and permissive are needed to allow replication
Determining the number of viruses in a sample (two methods)
• count physical virus particles (and their components)
(e.g. EM image or qPCR/digital PCR to count copies of the viral genome in a sample)
• count infectious virus particles (not all virus particles are infectious!*)
For each virus family there is a particle-to-PFU ratio = # physical virus particles / # infectious viral particles
(very different between viruses but for most viruses 1 infectious particle of the 100 virus particles produced in a cell)
--> sometimes 1 in 2 meaning that almost all particles are infectious and for some 1 in 10 000 viral particles
genome detection vs. infectious particles: detecting genomes or viral proteins does not give information about the
amount of infectious virus (e.g. Covid PCR: you have viral RNA in your body but you don’t know whether it is still infectious)
*Bacteriophages are very efficient (a single particle can initiate infection) but most eukaryotic animal viruses are less successful
--> defective and damaged virus particles / lethal mutations (in RNA viruses) / failure to complete replication cycle
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, Measuring infectious particles (done by infecting cells)
1. Plaque assay (plaques formed due to virus infection)
Used to measure the viral titer: make a 10-fold dilution series and add some of the diluted virus solution on cells
--> count plaques = holes in the cell monolayer, each originating from one infected cell (one infectious viral particle)
(the virus infects one cell and spreads to neighboring cells, destroying all these cells and thus creating a hole or plaque)
! number of plaque-forming units (infectious particles) per mL = number of plaques x dilution factor / volume added to cells
- if the virus stock is not sufficiently diluted, you will have too many infected cells so you won’t be able to count the plaques
- if the virus stock is diluted too much, it is not trustworthy
2. Endpoint dilution assay (assay developed because not all viruses form plaques)
Infect multiple wells for every dilution (keep on diluting the virus stock until it is a homeopathic dilution (so no virus particle present anymore)
and look in wells if there is infection or not (do you see CPE): the higher the dilution, the less infected wells you have
- graph: x-as = virus dilution and y-as = percentage of infected wells
- find TCID50 = value where 50% of your wells are infected
--> if you inoculate 10 wells with this amount of virus, 5 of them will be infected and 5 not
3. Multiplicity of infection (MOI) = number of infectious particles added per cell in a cell culture = PFU / number of cells
Based on the MOI, you can predict whether all cells will be infected or not
(you need a specific MOI to have almost all cells infected --> it never reaches 100% infection)
e.g. MOI= 10: 107 virus particles added per 106 cells --> you will probably infect 95% of you cells
! MOI ≠ the number of infectious particles that each cell receives --> infection of cells depends on
the random collision of virus particles with cells, so not all susceptible cells are infected when virus is added:
- some cells receive no virus and remain uninfected
- other cells receive 1 or more particles
e.g. MOI = 1 when you add 10 million virus particles to 10 million cells --> theoretically one infectious virus particle for each
cell, but some cells will be infected with 2 or 3 particles and other cells won’t be infected, so it does not mean you will infect
all cells (usually around 50-60% of infected cells)
Measuring physical virus particles
1. Hemagglutination assay (HA) and Hemagglutination inhibition assay (HIA) --> simple and inexpensive
Principle: sialic acid receptors on red blood cells bind to hemagglutinin glycoprotein found on the surface of several viruses
--> classis assay for Influenza viruses (does not work for all viruses since the virus needs to express agglutinins)
(hemagglutination = aggregating of red blood cells in the presence of agglutinins that bind sialic acid on the surface or RBCs)
- no hemagglutination = red dot = concentrated RBCs dropped down in the round-bottomed well due to gravity
- hemagglutination = viruses can bind to the surface of RBCs --> RBCs crosslinked by virus particles fall down
but not to the lowest point so they remain spread across the rounded plate (red lattice)
Hemagglutination inhibition assay to measure the presence of Ab:
if Ab recognize the virus, they will bind the virus so the virus can no longer bind RBCs --> inhibition of HA = red dot
2. Electron microscopy (no longer used for diagnosis because it’s too difficult and expensive but it is used for fundamental
research: to understand the virus structure and to see where virus particles are present in infected cells)
3. Measuring viral enzyme activity --> see whether a specific viral enzyme is present or not (e.g. reverse transcriptase)
4. Serology
• Direct: viral antigen detection (e.g. western blot to detect (viral) proteins)
• Indirect: antiviral antibody detection (IIFT: Indirect Immuno Fluorescence Test or Lateral flow tests (vgl zelftest COVID))
5. Viral genome (nucleic acid) detection by polymerase chain reaction (PCR)
- DNA viruses: regular PCR
- RNA viruses: RT-PCR (reverse transcriptase and then PCR)
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