1 Intro & mosquito-borne infections (incl. diagnostics)
1.1 Virus replication (+) RNA virus (poliovirus)
Poliovirus is a member of the Enterovirus genus, it can cause childhood paralysis and one of the best studied viruses at the molecular level.
It is a virus that is transmitted by an oral-faecal route and is vaccine preventable.
1. What type of viral genome does poliovirus have?
Positive sense, ss-RNA, non-segmented 8. How does poliovirus ensure translation of its viral
RNA by the host ribosome?
2. In an infected cell, what type of viral RNAs can you IRES element translation initiation factors bind to Vpg cap
find? independent translation
(-), (+), dsRNA, ssRNA
9. What could be a benefit of this approach?
3. What structure do cellular mRNAs have at their 5’ The benefit of initiating translation without a cap is that the
end? virus for instance could encode a protein which targets cellular
5’cap mRNAs at their cap. This way, only viral mRNAs are translated
and no cellular mRNAs
4. What structures does poliovirus have at the 5’ end?
VPG protein
10. Cellular mRNAs usually encode a single protein; the
(single) viral RNA of poliovirus encodes 11 proteins.
5. What structure do cellular mRNAs have at their 3’
What strategy does the virus use to achieve this?
end?
Polyprotein processing
Poly(A) tail
Poliovirus was the first (+) RNA virus for which an ‘infectious
6. What structures does poliovirus have at the 3’ end?
clone’ was made (nearly 40 years ago), a very important tool to
3’OH group and poly(A) tail
study functions of viral proteins and the effects of specific
mutations in viral genome.
7. What are the functions of the 5’ and 3’ structures on
cellular mRNA?
Stability of the RNA recruits poly(A) protein recruit
translation initiation factors recruit small ribosomal subunit
recruit large ribosomal subunit
1.2 Virus diagnostics
1. Virus diagnostics making use of assessing the samples. Cq values are inverse to the amount of target nucleic
cytopathic effect (CPE) in cell culture is not routinely acid that is in your sample, and correlate to the number of
used in laboratory diagnostics. Why is this the case? target copies in your sample. Lower Cq values (typically below
CPE is more laborious and has a long waiting time from sample 29 cycles) indicate high amounts of target sequence. Higher
collection to results. Virus identification is also often not Cq values (above 38 cycles) mean lower amounts of your target
possible. nucleic acid. High Cq values can also indicate problems with the
target or the PCR set-up, as outlined later in the pitfalls section
of this article.
2. What is the purpose of adding a semisolid agarose
matrix on top of the cell monolayer during a plaque
The more DNA in the sample, the lower the Ct sample. Ct says
assay?
how fast you reach a threshold.
The increased viscosity ensures that each infected cell only
infects its immediate neighbours. Diffusion is limited. 5. What do the secondary antibodies in an antibody
It prevents virus particles from freely floating through the cell ELISA recognize? The Fc part of antibodies
culture dish.
3. What is a prerecruitment for a virus to be plaqued?
Viruses that acutely affect cells and cause high CPE
4. Fluorescence and Ct values are readouts of a
quantitative PCR. How do they relate to the amount
of DNA in the sample?
Ct values are the same as Cq values. The Cq value or cycle
quantification value is the PCR cycle number at which your
sample’s reaction curve intersects the threshold line. This value
tells how many cycles it took to detect a real signal from your
1.1 Virus replication (+) RNA virus (poliovirus)
Poliovirus is a member of the Enterovirus genus, it can cause childhood paralysis and one of the best studied viruses at the molecular level.
It is a virus that is transmitted by an oral-faecal route and is vaccine preventable.
1. What type of viral genome does poliovirus have?
Positive sense, ss-RNA, non-segmented 8. How does poliovirus ensure translation of its viral
RNA by the host ribosome?
2. In an infected cell, what type of viral RNAs can you IRES element translation initiation factors bind to Vpg cap
find? independent translation
(-), (+), dsRNA, ssRNA
9. What could be a benefit of this approach?
3. What structure do cellular mRNAs have at their 5’ The benefit of initiating translation without a cap is that the
end? virus for instance could encode a protein which targets cellular
5’cap mRNAs at their cap. This way, only viral mRNAs are translated
and no cellular mRNAs
4. What structures does poliovirus have at the 5’ end?
VPG protein
10. Cellular mRNAs usually encode a single protein; the
(single) viral RNA of poliovirus encodes 11 proteins.
5. What structure do cellular mRNAs have at their 3’
What strategy does the virus use to achieve this?
end?
Polyprotein processing
Poly(A) tail
Poliovirus was the first (+) RNA virus for which an ‘infectious
6. What structures does poliovirus have at the 3’ end?
clone’ was made (nearly 40 years ago), a very important tool to
3’OH group and poly(A) tail
study functions of viral proteins and the effects of specific
mutations in viral genome.
7. What are the functions of the 5’ and 3’ structures on
cellular mRNA?
Stability of the RNA recruits poly(A) protein recruit
translation initiation factors recruit small ribosomal subunit
recruit large ribosomal subunit
1.2 Virus diagnostics
1. Virus diagnostics making use of assessing the samples. Cq values are inverse to the amount of target nucleic
cytopathic effect (CPE) in cell culture is not routinely acid that is in your sample, and correlate to the number of
used in laboratory diagnostics. Why is this the case? target copies in your sample. Lower Cq values (typically below
CPE is more laborious and has a long waiting time from sample 29 cycles) indicate high amounts of target sequence. Higher
collection to results. Virus identification is also often not Cq values (above 38 cycles) mean lower amounts of your target
possible. nucleic acid. High Cq values can also indicate problems with the
target or the PCR set-up, as outlined later in the pitfalls section
of this article.
2. What is the purpose of adding a semisolid agarose
matrix on top of the cell monolayer during a plaque
The more DNA in the sample, the lower the Ct sample. Ct says
assay?
how fast you reach a threshold.
The increased viscosity ensures that each infected cell only
infects its immediate neighbours. Diffusion is limited. 5. What do the secondary antibodies in an antibody
It prevents virus particles from freely floating through the cell ELISA recognize? The Fc part of antibodies
culture dish.
3. What is a prerecruitment for a virus to be plaqued?
Viruses that acutely affect cells and cause high CPE
4. Fluorescence and Ct values are readouts of a
quantitative PCR. How do they relate to the amount
of DNA in the sample?
Ct values are the same as Cq values. The Cq value or cycle
quantification value is the PCR cycle number at which your
sample’s reaction curve intersects the threshold line. This value
tells how many cycles it took to detect a real signal from your
