Lecture 1. Viruses background, classification and principles
What is a virus?:
• Small, obligatory intracellular, infectious agents
• Replication only in the host (in other cells)
• Fully dependent on a host for energy metabolism, lipid biogenesis and protein synthesis
= Obligate intracellular parasites or symbionts that possess their own genomes encoding
information required for virus reproduction and, hence, a degree of autonomy from the host
genetic system, but do not encode a complete translation system or a complete membrane aparatus s a s e r
l
Virus replication cycle:
1) Virus particle is binding to a receptor on the host cell
2) Entry/penetration of the virus particle
3) Uncoating of the virus: the nucleic acids can enter the cytoplasm
4) Replication of the virus: more DNA/RNA than the initial amounts
5) Assembling in new virus particles: proteins, capsid proteins and nucleic acids come together to make
virus particles
6) Releasing/leaving the cell:
Budding through the membrane (this acquires an envelope)
Cytolysis: the virus kills te host cell and the virus particles are released
Virus classification:
• All viruses must make mRNA that can be read by host ribosomes -
• More than thousand viruses: to organize these viral genomes, there are 7 genome types (7 groups)
Baltimore classification
Baltimore classification:
I: dsDNA viruses -3 HBV Herpes
,
Needs to make a mRNA that can be read by host ribosomes to make a viral protein
Transcribe their DNA into mRNA
II: ssDNA viruses
III: dsRNA viruses
IV: Positive strand ssRNA viruses - dengue Zika covid
,
s
,
Host ribosome recognizes the RNA directly and translate it
Directly read by the ribosome to make proteins
Messenger RNA strand
influenza RSV
l
V: Negative strand ssRNA viruses -
s
Need to make a positive strand (with a viral polymerase) before the ribosome can recognize it
,
and can translate it into proteins
Has the reverse complement RNA in the virus particle
VI: Retroviruses ~> HIV
VII: Gapped dsDNA viruses
Positive vs. negative strand RNA viruses - replication:
• Positive strand:
Directly translated to a protein m a m n
OR
Transcribed twice (+ to - to +) to make progeny + RNA to assemble into
new viruses
• Negative strand:
Transcribed to + strand RNA to translate to a protein
OR
Transcribed twice ( - to + to -) to make a progeny - RNA to assemble into
new viruses
mo
Virus classification - on physical characteristics:
• Nature (and sequence) of nucleic acid in virion
RNA vs DNA
ss or ds
Polarity (+ or -)
Segmented or non-segmented
• Symmetry of capsid
Helical or icosahedral {
• Presence/absence of lipid membrane
round)
Enveloped or naked/non-enveloped
• Dimensions of virion and capsid
Virus taxonomy
• Classification of viruses
• Viruses within the same family or genes share the same replication strategy, but don’t transmit the same way and don’t cause the same
diseases!
Doesn’t explain the tropism or pathogenesis
,Lecture 2. Detection of viruses and methods for virus diagnostics
Detection of viruses:
• Diagnosis for patients with a suspected virus infection
• Fundamental virology/immunology research
• Epidemiological research
• Surveillance
Direct and indirect diagnostics:
• Direct (measuring the pathogen)
Detection of the whole microorganism (infectious particle)
Culture based techniques & (electron) microscopy
Detection of structural components (viral proteins or genetic material)
Antigen ELISA, fluorescence microscopy & lateral flow assay
Detection of products (toxics from bacteria and/or fungi)
PCR & sequencing
• Indirect (serology)
Detection of antibodies targeting microorganisms in serum of a patient
Antibody ELISA & agglutination test
Direct diagnostics - microscopy:
• The biggest viruses are detectable with light microscopy, but light microscopy is for most of the viruses impossible
• Electron microscopy: not used in diagnostics and this method is not discriminative
Direct diagnostics - culture based detection method:
• Detect the damage from the virus to cells: cytopathic effect (CPE)
Infection result in structural changes in a monolayer of susceptible cells
Cytopathic effect (CPE):
I) Virus infects susceptible cells
II) Replication of the virus (genome replication and amplification of virus particles)
III) Release of new virus particles
IV) Infection of new cells
V) Structural changes -> cells die or fuses together to form giant cells (syncytua)
Direct diagnostics - quantification of the infectious virus particles:
• Plaque assay
A local area in which cells have died due to virus infection
A plaque is the result of a single initial virus infection event
Infect one single cell -> neighboring cells are infected -> this creates a local area where cells are going to die
Dilute the virus far enough to get a single infection on the plate and to calculate the amount of virus in the original sample
Agarose matrix on top to prevent freely floating of virus particles through the cell culture dish
• End point dilution
Used for viruses that don’t form plaques
Making a dilution in a well-plate and each well is scored
The end point is the dilution of the virus at which 50% of the cell cultures are infected
Direct diagnostics - detection of genetic material:
• Q(PCR)
Sensitive and specific
Quantification is possible (Ct-value)
Fast (couple of hours)
• Next generation sequencing
Detect small differences in the genome
Identify the unexpected
Taqman QPCR
• Measure the amount of DNA after every cycle
• The more DNA double strands, the more fluorescence to detect and measure
• Probe with a fluorescence dye and a quencher
The quencher is quenching the fluorescence molecule in an intact probe
Culture based techniques vs PCR:
• Culture bias technique
+ = detection of infectious particles and gives the chance to find the ‘unexpected’
- = more laborious, long time waiting from sample collection to result, and virus identification is not always possible
• PCR
+ = high sensitivity, high specificity and relatively short time from sample collection to result
- = relatively expensive, (too) high sensitivity and the analysis is biased towards expected viruses
,Direct diagnosis- antigen based detection methods:
• Antibodies that bind to the virus protein
• Antibody-antigen interaction with labelling
Direct antigen test: labelled antibodies that binds to the specific virus
antigens -> EXPENSIVE
Indirect antigen test: unlabelled antibodies that binds to the specific virus
antigen (primary antibody) + a secondary labelled antibody that recognize
the primary antibody (as an antigen)
Direct diagnosis - Enzyme-Linked Immuno Sorbent Assay (ELISA) to detect
antigens:
• Measuring antigens
I) Binding of an antibody to the solid phase
II) Add unknown antigen (sample)
III) Detect bound antigen with labelled second antibody
Indirect diagnostics:
• AB ELISA
• Agglutination
Antibody response to infection:
• Infection -> antibody response?
Seroconversion: the development of a specific antibody response as a result
of immunization
• Initial antibody response: IgM mediated, but there is a isotyp switch from IgM to
IgG after a few weeks
IgG is detectable for many months, years or even lifelong
Indirect diagnostics - AB ELISA:
I) Bind antigen to solid phase
II) Add unknown antibody
III) Detect bound antibody with labelled antibody
Indirect diagnostics - Agglutination assay:
• Test whether immune complexes are formed between antibodies present in a
sample and latex beads that are covered with a specific antigen
, Lecture 3. Arboviruses - virus replication and transmission
determines pathogenesis & transmission
Virus tropism r
• Which tissues and organs are infected is largely determined by the presence of susceptible cells (cells with a functional receptor for the
virus)
• Cellular tropism: the virus replicates in one cell type, but not in another cell type (HIV infects macrophages and not neurons)
• Tissue tropism: the virus replicates in a particular tissue or organ, but not in another tissue or organ (influenza infects lung tissue and not
brain tissue)
• Host tropism: the virus replicates in one host specie, but not in other species (myxoma virus infects rabbits and not humans)
Transmission cycle
• Mosquitos are not flying needles: there is active replication in the vertebrate host AND in the mosquito vector
• Extrinsic incubation period: the time between taking an infectious bloodmeal and being able to transmit the virus
• The virus enters the mosquito through the blood of an infected person
• The infectious particles spread through the foregut to the anterior and posterior midgut
• Replication takes place in the posterior midgut, where enough virions are produced, so they
are passed from mosquitos to uninfected persons through the salivary glands by pricking
Barriers for the infection
• Midgut infection barrier (MIB): factors that impede virus infection of midgut epithelium
Entering of the epithelium cells is impeded
• Midgut escape barrier (MEB): factors that impede release of viruses from midgut epithelium
Leaving from the epithelium cells is impeded
• Salivary gland infection barrier & salivary gland escape barrier are similar barriers of the midgut barriers
In summary:
The virus enters the arthropod after an infected blood meal, it passes through the gut wall to reach the salivary gland, where replication takes
place: in the midgut. So, the first thing that needs to happen to the virus is to leave the blood and infect the epithelial cells of the midgut.
If the virus is not able to do so it cannot pass the so-called Midgut infection Barrier (MIB). The second thing is to leave the epithelial cells on
the other site. If they are not able to do so they cannot pass the so-called Midgut Escape Barrier (MEB).
Vector competence and vectorial capacity
• Competence: the ability of a mosquito to orally acquire, maintain and transmit a pathogen to a next vertebrate
• Capacity: the combination of abiotic and mosquito intrinsic factors that determine the potential to transmit a given pathogen
• Extrinsic- and intrinsic factors can influence the transmission:
Extrinsic: temperature, population density and vector-host contact
Intrinsic: immunity, barriers and genetics
Transmission cycles
• Enzootic cycle: most viruses are not directly circulating between mosquitos and humans: wild animals-mosquitos-humans
West Nile virus: circulated between mosquitos and birds and nowadays between mosquitos and humans
These viruses have an animal reservoir, which means that a vaccination can’t stop the virus circulation
Humans are dead-end hosts: the virus level in blood is to low for further transmission
• Urban epidemic cycle: direct transmission from humans to humans
