Self-study assignments Infectious diseases
Self-study assignment 1 Virus replication (+) RNA virus (poliovirus)
1. What type of viral genome does poliovirus have?
Linear positive sense ssRNA non-segmented (single stranded RNA). A polymerase translated from viral
mRNA produces negative sense RNA from the positive sense template, which is then transcribed
repeatedly into more positive strands. Further cycles of transcription then occur, resulting in the
production of very large numbers of positive strands, which are packaged into new particles using
structural proteins translated earlier from mRNA.
2. In an infected cell, what type of viral RNAs can you find?
Double stranded RNA (dsRNA), positive sense ssRNA, negative sense ssRNA, positive sense mRNA.
3. What structure do cellular mRNAs have at their 5’ end?
5’ cap/RNA cap. A modified guanine (G) nucleotide.
4. What structures does poliovirus have at the 5’ end?
Type I internal ribosome entry site (IRES), viral protein (VPg)
5. What structure do cellular mRNAs have at their 3’ end?
3’ Poly-A tail
6. What structures does poliovirus have at the 3’ end?
3’ Poly-A tail
7. What are the functions of the 5’ and 3’ structures on cellular mRNA?
The 5’ cap protects the transcript from being broken down. It also helps the ribosome attach to the
mRNA and start reading it to make a protein.
The 3’ poly-A tail makes the RNA molecule more stable and prevents its degradation. Additionally, the
poly-A tail allows the mature messenger RNA molecule to be exported from the nucleus and translated
into a protein by ribosomes in the cytoplasm.
, 8. How does poliovirus ensure translation of its viral RNA by the host ribosome?
The 5' VPg can be cleaved off the genomic RNA by host TBP2, also called "unlinkase" . This creates a pool
genomes with a 5'pUp used for translation/replication.
Poly-A tail inhibit the cell's poly-A binding protein (PABPC1) in order to emphasize their own genes'
expression over the host cell's.
- Cap independent translation
- IRES element
- Vpg has nothing to do with translational initiation
9. What could be a benefit of this approach?
Polioviruses shut down cap-dependent translation in order to allow more efficient IRES-dependent
translation. It allows ribosome recruitment under conditions where cap-dependent protein synthesis is
severely repressed why useful? to interfere with the host translational machinery thereby shutting
of host gene expression. The benefit of initiating translation without a cap is that the virus for instance
could encode a protein which targets cellular mRNAs at their cap. This way, only viral mRNAs are
translated and no cellular mRNAs.
10. Cellular mRNAs usually encode a single protein; the (single) viral RNA of poliovirus encodes 11
proteins. What strategy does the virus use to achieve this?
The polyprotein is initially processed by the viral proteases into three precursor proteins, P1, P2, and P3.
Precursor P1 is then proteolytically cleaved to yield the structural proteins. Precursors P2 and P3 are
processed into replicase, VPg, and a number of proteins that modify the host cell, ultimately leading to
cell lysis.
In viruses where the genome is a single nucleic acid molecule, translation produces a large
multifunctional protein, a polyprotein, which is then cleaved enzymatically to produce a number of
distinct proteins.
Viral proteases may also cleave cellular proteins to modulate the host. Polyprotein processing.
Self-study Virus diagnostics
1. Virus diagnostics making use of assessing the cytopathic effect (CPE) in cell culture is not
routinely used in laboratory diagnostics. Why is this the case?
Long waiting time from sample collection to result. Virus identification is often not possible.
2. What is the purpose of adding a semisolid agarose matrix on top of the cell monolayer during
a plaque assay?
The viral particles cannot freely float through the medium. They have to infect the neighboring cells
themselves.
, Prerequisite for viruses to be able to use plaque assay they need to be causing cell death
3. Fluorescence and Ct values are readouts of a quantitative PCR. How do they relate to the
amount of DNA in the sample?
Maximum fluorescence plateau phase. In qPCR-ct value is defined as the number of amplification
cycles required to reach a fixed signal threshold. Lower ct value higher DNA amount in sample.
4. What do the secondary antibodies in an antibody ELISA recognize?
It recognizes the Fc part of the antibody. This is the defense antibody against the viral particles.
5. Which diagnostic technique is based on the detection of virus induced cytopathic effect (CPE)
in cell culture?
Plaque assay and end-point dilution
6. A technician in a diagnostics laboratory has been asked to test whether a patient has had a
SARS-CoV2 making use of an antibody ELISA. Which type of sample is needed for this analysis?
Blood serum
Self-study assignment 1 Virus replication (+) RNA virus (poliovirus)
1. What type of viral genome does poliovirus have?
Linear positive sense ssRNA non-segmented (single stranded RNA). A polymerase translated from viral
mRNA produces negative sense RNA from the positive sense template, which is then transcribed
repeatedly into more positive strands. Further cycles of transcription then occur, resulting in the
production of very large numbers of positive strands, which are packaged into new particles using
structural proteins translated earlier from mRNA.
2. In an infected cell, what type of viral RNAs can you find?
Double stranded RNA (dsRNA), positive sense ssRNA, negative sense ssRNA, positive sense mRNA.
3. What structure do cellular mRNAs have at their 5’ end?
5’ cap/RNA cap. A modified guanine (G) nucleotide.
4. What structures does poliovirus have at the 5’ end?
Type I internal ribosome entry site (IRES), viral protein (VPg)
5. What structure do cellular mRNAs have at their 3’ end?
3’ Poly-A tail
6. What structures does poliovirus have at the 3’ end?
3’ Poly-A tail
7. What are the functions of the 5’ and 3’ structures on cellular mRNA?
The 5’ cap protects the transcript from being broken down. It also helps the ribosome attach to the
mRNA and start reading it to make a protein.
The 3’ poly-A tail makes the RNA molecule more stable and prevents its degradation. Additionally, the
poly-A tail allows the mature messenger RNA molecule to be exported from the nucleus and translated
into a protein by ribosomes in the cytoplasm.
, 8. How does poliovirus ensure translation of its viral RNA by the host ribosome?
The 5' VPg can be cleaved off the genomic RNA by host TBP2, also called "unlinkase" . This creates a pool
genomes with a 5'pUp used for translation/replication.
Poly-A tail inhibit the cell's poly-A binding protein (PABPC1) in order to emphasize their own genes'
expression over the host cell's.
- Cap independent translation
- IRES element
- Vpg has nothing to do with translational initiation
9. What could be a benefit of this approach?
Polioviruses shut down cap-dependent translation in order to allow more efficient IRES-dependent
translation. It allows ribosome recruitment under conditions where cap-dependent protein synthesis is
severely repressed why useful? to interfere with the host translational machinery thereby shutting
of host gene expression. The benefit of initiating translation without a cap is that the virus for instance
could encode a protein which targets cellular mRNAs at their cap. This way, only viral mRNAs are
translated and no cellular mRNAs.
10. Cellular mRNAs usually encode a single protein; the (single) viral RNA of poliovirus encodes 11
proteins. What strategy does the virus use to achieve this?
The polyprotein is initially processed by the viral proteases into three precursor proteins, P1, P2, and P3.
Precursor P1 is then proteolytically cleaved to yield the structural proteins. Precursors P2 and P3 are
processed into replicase, VPg, and a number of proteins that modify the host cell, ultimately leading to
cell lysis.
In viruses where the genome is a single nucleic acid molecule, translation produces a large
multifunctional protein, a polyprotein, which is then cleaved enzymatically to produce a number of
distinct proteins.
Viral proteases may also cleave cellular proteins to modulate the host. Polyprotein processing.
Self-study Virus diagnostics
1. Virus diagnostics making use of assessing the cytopathic effect (CPE) in cell culture is not
routinely used in laboratory diagnostics. Why is this the case?
Long waiting time from sample collection to result. Virus identification is often not possible.
2. What is the purpose of adding a semisolid agarose matrix on top of the cell monolayer during
a plaque assay?
The viral particles cannot freely float through the medium. They have to infect the neighboring cells
themselves.
, Prerequisite for viruses to be able to use plaque assay they need to be causing cell death
3. Fluorescence and Ct values are readouts of a quantitative PCR. How do they relate to the
amount of DNA in the sample?
Maximum fluorescence plateau phase. In qPCR-ct value is defined as the number of amplification
cycles required to reach a fixed signal threshold. Lower ct value higher DNA amount in sample.
4. What do the secondary antibodies in an antibody ELISA recognize?
It recognizes the Fc part of the antibody. This is the defense antibody against the viral particles.
5. Which diagnostic technique is based on the detection of virus induced cytopathic effect (CPE)
in cell culture?
Plaque assay and end-point dilution
6. A technician in a diagnostics laboratory has been asked to test whether a patient has had a
SARS-CoV2 making use of an antibody ELISA. Which type of sample is needed for this analysis?
Blood serum
