Altklausuren Flashcards
1
Q
Wer widerlegte die endgültige Ursprungstheorie? (Name) Mittelchen Technik?
A
Louis Pasteur -> Swan Neck bottle
2
Q
a) Nennen Sie die Kochechten Postulate
b) Welche Probleme treten bei Viren im Vergleich mit Bakterien bei den Postulaten auf?
c) Nennen Sie einen Virus, bei dem der Nachweis besonders problematisch war. Warum?
A
a) 1. Erreger muss sich in allen erkrankten nicht aber in Gesunden nachweisen lassen.
2. Erreger muss sich in Reinkultur Anzüchten lassen.
3. Erreger muss dieselbe Krankheit auch in anderen auslösen.
4. Erreger kann von den neu Erkrankten wieder isoliert werden.
b) Viren sind obligat intrazellulär und lassen sich nicht ohne spezifisch passende Zellen Anzüchten.
c) Hepatitis C
3
Q
Actinomycin inhibiert
A
- RNA Polymerase II
- Influenza A
- Herpesvieren
4
Q
Wieso liefern die Methoden zur Influenzaquantifizierung unterschiedliche Ergebnisse?
A
Methoden liefern unterschiedliche Ergebnisse, da sie alle Vieren, nur infektiöse Viren oder Untereinheiten (HA) nachweisen (Unterschied egg-pfui -> egg lässt sich effizienter infizieren (ganzer Organismus))
5
Q
Was war das erste identifizierte Virus?
A
Tabbak-Mosaic (Gelbfieber war das erste humane Virus)
6
Q
Was war die entscheidende Eigenschaft, die die Abtrennung von Viren von Bakterien ermöglichte?
A
Ultrafiltrierbarkeit
7
Q
Nennen Sie die Virusfamilie und -genera des Blauzungenvirus
A
Familie: Reoviridae
Genus: Orbivirus
8
Q
Welcher Blauzungen-Serotyp war für den aktuellen Ausbruch verantwortlich?
A
Serotyp 8 -> Impfung vorhanden; Serotyp 6 (jetzt hier Weden illegaler Impfung)
9
Q
Beschreiben Sie die Virionmorphologie vom Blauzungenvirus mit wenigen aussagekräftigen Stichpunkten
A
- kubisch
- behüllt
- 3 layer (Virion, ISVP, Core)
10
Q
Beschreiben Sie den Aufbau des Genom des Blauzungenvirus
A
10-12 lineare dsRNA Segmente
11
Q
Hat die Impfung gegen BTV geholfen, das Virus in Deutschland in den Griff zu bekommen? Reicht dies, um das Virus endgültig auszurotten?
A
Typ 8: ja im Griff, Virus aber dadurch nicht ausgerottet (Impfung verringert nur Virämie)
12
Q
Was versteht man unter einem Viriod? Nennen Sie ein Beispiel
A
infektiöse RNA ohne codierte Proteine
13
Q
Kann man gegen Viroide impfen? Wenn ja, wie?
A
ja, mit “Impfung” mit virulenten Formen
14
Q
Nennen Sie drei cytopathische Effekte und jeweils ein Virus, das diesen Effekt hervorruft
A
Lyse: Polio
Transformation: Retro
riesige Zellen: Cytomegalo
Synzytien: Masern
inclusion bodies: Adeno
15
Q
Which instrument/method enabled Iwanovski (1892) to differentiate viruses from the already known bacteria?
A
ceramic or chamberlain filter (Viren gehen durch; Bakterien bleiben hängen)
16
Q
To which family and genus (correct phylogenetic names!) does Yellow Fever virus belong?
A
Familie: Flaviviridae
Genus: Flavivirus
17
Q
Specify the structure of the genome and the virion of the Yellow Fever Virus
A
Virion: behüllt und kubisch (ikosaeder), ca 50 nm
Genom: linear, ss (+)-strand RNA, ca 10-12 kb, 1 ORF
18
Q
When, how (method) and from whom was a vaccine (name of vaccine) for Yellow Fever Virus developed? Catchwords only
A
1930 Max Teiler 17D? attenuation in Mäusen
19
Q
How can influenza virus particles be counted? Give short description of the four different methods and state what each of them actually measures!
A
Electron microscopy (EM): man zählt direkt alle Viren (übers Anhängen elektrodichter Partikel)
quantal infectivity assay in eggs: man zählt alle infektiösen Viren ( sichtbar werden z.B. Hemorrhages)
quantitation by plaque forming units (pfu): man zählt alle infektiösen Viren (immer wenn ein Loch im Zellrasen entsteht, wurde dieses durch ein infektiöses Virus verursacht)
Hemaglutinin assay (HA): hier zählt man Partikel die HAs enthalten
20
Q
What is a viroid (definition)?
A
infektiöse RNA, die keine Proteine codiert
21
Q
Give an example of a viroid with economical relevance
A
Crysanthenum stunt viroid
22
Q
Is protective vaccination against viroids possible? if yes, how?
A
möglich durch “Impfung” mit virulenter Form
23
Q
Erkläre kurz, warum Polioviren nicht von Antimycin D und warum Influenza Viren von Antimycin D inhaliert werden
A
Polio: hat seine eigene Polymerase und repliziert im Cytoplasma
Influenza: hat zwar eigene Polymerase, repliziert aber im Kern und nutzt zelluläre Präsentation-mRNA für das Cap Snatching
24
Q
Ankreuzaufgabe
A
- Nackte (+) Strang RNA Viren sind infektiös
- Visions of negative-strand RNA viruses contain a viral RdRp
25
Which molecules may contain "kissing-loop interactions" and "pseudoknots"?
RNA
26
In which regulatory mechanism is the "pseudo knot" of functional importance?
ribosomal frameshifting
27
Name two viruses from different families which use ribosomal frameshifting
SARS-Coronavirus
die meisten Retroviren
28
What is a CRRE-element and what is it function in the replication of Poliovirus?
- intragenomic cis-active replication element
- das Vpg bindet zusammen mit der 3D-Pol an das Credo und wird dort uridinyliert, woraufhin es im Komplex mit der polymerase an das Poly-A binden kann
29
Which mechanisms of posttranscriptional translational regulation does Sendaivirus use? Name and describe (shortly!) the mechanisms
ribosome shunting
durch Sekundärstrukturen der RNA wird der gesamte Polymerasekomplex woanders hin gebunden und startet erst dort die Suche nach dem AUG
leaky scanning
kein Start am 1.AUG, da dieses in einem schlechten "Kontext" steht und somit gar nicht als solches erkannt wird -> nächstes AUG = Start cotranscriptionales RNA editing (Insertionen, Basenaustausche)
Insertionen: RNA-Pol pausiert in der Nähe einer U-C-junction nachdem 2C eingebaut wurden -> die Pol und gebundenen mRNA rutschen ein Stück zurück -> zusätzliche Nucleotide werden eingebaut (1-2)
30
List 4 viruses or virus-like agents involved in hepatitis in humans. Also state to which virus family they belong.
HAV: Picornaviridae
HBV: Hepadnaviridae
HCV: Flaviviridae
HDV: nicht klassifiziert (ist ein Virosoid und HBV-abhängig)
31
Explain the transcription mechanism of the members of the family Coronaviridae.
Nidovirales -> nested set -> 3' coterminale mRNAs mit identischem 5' werden generiert: zwischen den einzelnen ORFs befinden sich TRS (transcription regulating sequence) -> vom Poly-A aus werden die (-)-Stränge transkribiert; die Synthese kann auch an den TRS abbrechen oder reinitiiert werden -> das endständige TRS bindet nach Abbruch ans leader-TRS (direkt nach dem Cap) und die (-)-Strang Synthese wird beendet (alle haben also dieselbe 5' Cap Region) -> von jeder mRNA wird immer nur das 5' proximale ORF translatiert
32
Ankreuzaufgabe Picornavirus
Picornaviruses possess a protein primer
33
What does the abbreviation ARBO virus stand for? Give one example (family, genus, species)
Arthropode borne virus
Familie: Reoviridae
Gattung: Orbivirus
Spezies: Blue-Tongue-Virus
34
Explain the mechanism "attenuation of transcription" for the negative strand RNA virus VSV.
Polymerase bindet ans 3' vom N-Gen, dieses wird transkribiert in den Ig's findet die Termination über 7U's (stottern und Polyadenylierung) statt -> die Polymerase ist nicht sehr prozessiv und Reinitiation findet somit nur in wenigen Fällen statt
somit werden die mRNAs in folgendem Mengenverhältnis hergestellt:
N > P > M > G > L
35
Influenzavirus Ankreuzaufgabe
- PB1 is the cap binding protein
- Viral proteins contain nuclear localization signal
35
Influenzavirus Ankreuzaufgabe
- PB1 is the cap binding protein
- Viral proteins contain nuclear localization signal
36
Influenzavirus Ankreuzaufgabe
- PB1 is the cap binding protein
- Viral proteins contain nuclear localization signal
37
Nennen Sie Kriterien nach denen Viren eingeteilt werden.
- Vorhandensein einer Lipidumhüllung
- Natur des viralen Genom (DNA oder RNA)
- Symmetrie des Kapsids
- Größe von Virion und Kapsid
38
Definieren Sie Serotyp und nennen Sie zwei Beispiele
- Variation innerhalb einer Subspezies, die mit serologischen Tests unterscheidbar sind
- Beispiel: Blue tongue disease -> Pathogen is very variable
38
Definieren Sie Serotyp und nennen Sie zwei Beispiele
- Variation innerhalb einer Subspezies, die mit serologischen Tests unterscheidbar sind
- Beispiel: Blue tongue disease -> Pathogen is very variable
39
Nennen Sie die Funktion von IRES-Strukturen
- allow binding of ribosomes without scanning
- mediate internal entry of the 40S ribosomal subunit on some eukaryotic and viral messenger RNAs upstream of a translation initiation codon
40
Welches Institut ist für die Klassifizierung von Viren zuständig (Abkürzung und voll ausgeschrieben)
ICTV: international committee on the taxonomy of Viruses
Virology Division of the International Union of Microbiological Societies
41
Aus welchem Tier stammt SARS?
Civet cat
42
Nennen Sie Rezeptorproteine von HCV, welche beim Eindringen in den Wirt beteiligt sind.
- CD81
- Low density lipoprotein (LDL) receptor
- Scavenger (SR-BI) receptor
- Claudin 1 (CLDN1)
- Occluding (OCDN)
43
Wofür steht die Abkürzung MOI, was wird damit gezählt?
= multiplicity of infection
- median number of infectious virus particles per cell
44
Nennen Sie Gründe, weswegen das Mimivirus ein Virus ist und kein intrazelluläres Bakterium
- capsid protein (double jelly-roll fold)
- ikosahedral strucutre
- eclipse phase ("disappearance") as part of life cycle
- absence of genes for ribosomes (but for aminoacyl tRNA synthetases), energy production or energy conversion
- phylogeny
45
Nennen Sie zwei Viren mit großen RNA-Genom (mit Genus und Familie!)
Nidovirales: Coronaviruses, Toroviruses, Androniviruses
45
Nennen Sie zwei Viren mit großen RNA-Genom (mit Genus und Familie!)
Nidovirales: Coronaviruses, Toroviruses, Androniviruses
45
Nennen Sie zwei Viren mit großen RNA-Genom (mit Genus und Familie!)
Nidovirales: Coronaviruses, Toroviruses, Androniviruses
46
Beschreiben Sie die Terminale Sequenz von Adenoviren!
- invertierte, terminale Repetitionen von 50-200 Basenpaare
47
a) Aus welchen Tieren und welchem Organ wurde Influenza das erste mal isoliert?
b) In welchem Produktionssystem wird Influenza vermehrt für die Vaccineherstellung?
a) Wasservögel
b) Hühnereier
48
Welches war die erste humane Zelllinie, welche konstant im Labor gewachsen ist?
HeLa Zellen
49
Nennen Sie drei verschiedene Formen des cytopathischen Effekts mit je einem Virus
1) Schwellung und Abrundung der Zellen mit nachfolgender Zytolyse
2) Loslösung vom Zellverband
3) Transformation von Zellen, z.B. Bildung mehrkerniger Riesenzellen (Synzytien)
4) Bildung von Einschlusskörperchen (Proteinaggregate bei exzessiver Proteinsynthese)
5) Vakuolisierung des Zytoplasmas
50
Definieren Sie Virusoid und nennen Sie ein Beispiel
- unvollständige ssRNA-Viren, denen Gene für wichtige Hüllproteine fehlen, weshalb sie für das Budding auf die Anwesenheit von Helferzellen angewiesen sind
- Beispiel: Hepatitis D
51
Wofür steht die Silbe Arbo in Arboviren? Geben Sie ein Beispiel an (Familie, Genus, Art)
- werden durch Anthropoiden übertragen
Familie: Flaviviridae
Genus: Flavivirus
Art: Dengue Virus
52
a) Was ist ein CRE-Element? Geben Sie den vollen Namen an.
b) Wo ist ungefähr im Poliovirus das CRE-Element lokalisiert?
c) Was ist die Funktion von einem CRE-Element im viralen Lebenszyklus?
a) cis-active replicative element
b) located within the poly protein-coding region
c) template for uridylation of VPN primers by 3Dpol
53
Was ist ein "RNA Repliken"? Was ist der Vorteil gegenüber dem ursprünglichen Virus, wenn man mit beiden im Labor arbeitet?
- DNA- oder RNA-Abschnitt, der ausgehend von einem Replikationsursprung an einem Stück repliziert wird
- liegt zwischen zwei Origins
- reduction of genome size to minimal set of genes required for RNA replication
54
Listen Sie drei Familien von Viren auf dessen Mitglieder Hemorrhagisches Fieber verursachen. Zu jeder Familie ein Virus.
- Flavivirus -> Dengue virus, Yellow fever virus
- Bunyaviruses -> Hantaan virus, Crimean-Congo hemorrhagic fever virus (CCHFV)
- Rhabdoviridae -> Bas-Congo Virus
55
Name three viruses from the genus Flavivirus
- Yellow fever virus
- Dengue virus
- Tick borne disease
56
At which membrane does the budding of retroviruses occur?
plasma membrane
57
List four forms of viral persistence, state the defining features and give for each one a viral example
1) Strategy of intrauterine infection
-> Acquired pathogen specific immun-tolerance/ no adaptive immune response
2) Strategy of suppression of the innate immune system
-> Nero and Erns: Block of IFN induction/innate immunity
3) Strategy of suppression of apoptosis/the innate immune system
-> Stringent regulation of NS3 release/RNA replication due to limiting cellular protease cofactor
-> Requirement for ncp biotype and absence of IFN response
4) High antigenic variability of the viral surface antigen
-> Reinfection of pregnant cows despite of previous BVDV infection
Beispiel: HCV
58
Name two viruses (virus and family) whose infection occurs via the gastro intestinal tract
- Caliciviridae -> Calicivirus
- Coronaviridae -> Coronavirus
59
What does the abbreviation "GALT" stand for?
gut associated lymphoid tissue
60
Explain shortly the terms and molecular basis of "antigenic shift" and "antigenic drift" in the context of the influenza virus system
Antigenic shift:
- missing accuracy of RNA polymerases
- RdRp without proofreading activity
- 1 mistake per 10^3-10^4 bases, accordingly 1-10 mistakes in each new genome
- Formation of quasispezies
Antigenic drift:
- segmentation allows reassortment
- confection with two related segmented viruses allows for reassortment
61
Name the two models for the mechanism of RNA recombination
1) "template switching": during viral RNA-replication
2) "breakage and ligation": independent of viral replication
62
Name two RNA viruses for which the process of RNA recombination is an essential step in the regular replication cycle? Explain by using catchwords
- Pestiviruses: insertions often code for cellular proteases
- noncp BVDV: cp variants may be generated by RNA recombination
63
(+)-strand RNA viruses restructure the intracellular membrane system of the host cell for their genome replication.
Name for each of the compartments listed below one virus which makes use of these membranes for genome replication
ER: HCV
Golgi: Kunjin virus (Flavivirus)
Lysosomen: Rubella virus
Mitochondrien: Flock house virus
64
Which of the hepatitis viruses frequently induces persistent infections?
HCV
(HBV)
65
Some humans have a genotype which protects them against infection by certain viruses. Name two human gene variations and the viruses against which they are protective as well as the putative underlying mechanisms.
HCV: Genetic variation in IL28B -> upstream region of IL28B gene = IFN-gamma3 = IFN class 3 molecule
SARS-CoV: Trim 55
66
What does the term "trans-infection" describe and in which virus system it was discovered?
- a cell "holds" a pathogen without getting infected (the pathogen does not enter the cell), only to pass it to another cell to infect it
- HIV-1?
67
Which alpha virus led to massive outbreaks in the human population since 2013? Name virus, main symptoms and route of transmission.
- Chikungunya Virus -> ss(+)RNA
- gehört zur Gruppe der Arboviren -> wir durch den Stich von Gliederfüßern übertragen (z.B. Mücken)
- verursacht Chikungunya Fieber
68
Denominate the genome of a herpes virus
- differential gene regualtion: immediate early, early, late genes
IRL: inverted region long
IRS: inverted region short
69
What are the two basic molecular models explaining the process of RNA recombination?
1) "template switching": during viral RNA-replication
2) "breakage and ligation": independent of viral replication
70
For which viruses RNA recombination events are part of their regular life cycle?
Pestiviruses: noncp BVDV --> cpBVDV
71
Explain the generation, the function/mode of action of the pr peptide during vision morphogenesis for members of the genus Flavivirus.
pr = proteolytic product
-> in the mature, infectious vision, the pr peptide is absent, and the virus undergoes membrane fusion in the endoscope at low pH
72
What is the FIP virus? Which disease is associated with this virus? What is unusual about this disease/pathogen?
FIP = Feline Infektiöse Perotonitis
- ansteckende Bauchfellentzündung
- virale Infektionskrankheit bei Katzen, die durch Mutation feiner Coronaviren verursacht wird (jeder Coronavirus kann pathogen werden)
- Infektion findet meist in den ersten Lebenswochen über persistent infizierte Tieren statt -> auf einen Reiz (z.B. Stress) hin, kann das Virus mutieren und schließlich das Krankheitsbild hervorrufen
-> besonders gefährdet sind Katzen, die ebenfalls mit der Feinen Legukose (FeLV) infiziert sind
73
What is the difference between pathogenicity and virulence of a germ?
pathogenicity = - quality or state of being pathogenic -> the potential ability to produce disease
- qualitative term -> "all or none" concept
virulence = - disease producing power of an organism
- degree of pathogenicity within a group or species
- term that quantifies pathogenicity
74
Name four mechanisms how to bend a membrane
- oligomers of viral membrane proteins induce membrane bending
CLUSTERING
- transmembrane proteins with inherent curvature inducing curvature in a membrane
MOTIF INSERTION
- insertion of a piece of a protein into one leaflet of the membrane induces curvature
BAR DOMAINS
- a BAR domain of a protein inducing and stabilizing the curvature of a membrane
SCAFFOLDING
- cage like structure of Cathrin
- when this structure forms around a membrane, it pulls the membrane into a tight curvature until eventual vesicle budding
CYTOSKELETON
- cytoskeletal structure inducing membrane curvature
- the inherent shape of a cell as controlled by its cytoskeleton requires that the bilayer membrane curve around it
CROWDING
-when a high local concentration of proteins are present on the membrane surface, membrane curvature can be induced
-> high protein concentration increases the likelihood of repulsions between proteins, therefore generates steric pressure between proteins. To relieve such pressure, lipid membrane has to bend in order to decrease protein repulsions.
75
What does the term "virological synapse" describe and which virus does use it?
- molecularly organized cellular junction that is similar in some aspects to immunological synapses
-> Herpes simplex virus (HSV)
-> Human immunodeficiency virus (HIV)
76
Name four viruses from different families (name virus and family) which can cause hemorrhagic fever.
Flaviviruses: Dengue, Yellow Fever
Bunyaviruses: Crimean-Congo hemorrhagic fever virus
Hantavirus
77
Which ARBO virus was recently introduced into the Americas and is suspected to cause severe brain damage in human fetuses? Name virus, family, genus and vector.
Zika Virus
Familie: Flaviviridae
Genus: Flavivirus
78
List three cellular sensors of double stranded RNA
- RIG-/- like receptors
- protein kinase R
- oligoadenylate synthases
- adenosine deaminases
79
What was the so called "Rabbitpox-Experiment"? What was the outcome and what could be learned?
- a highly virulent vaccinia virus strain, has caused outbreaks of a lethal pox disease in colonies of laboratory rabbits in Utrecht and New York
80
Definition "permissive" and "susceptible"
A cell is PERMISSIVE for a virus when genome replication is possible.
A cell is susceptible for a virus when the cell can be infected.
81
What is a viroid (definition)?
- small (200-400 nt), circular RNA
- self-complementary, rod-like secondary structure
- encode no protein
- no capsid or envelope
- replicate in nucleus or chloroplast
- cause severe disease in plant
- 20 species, 2 families
- replication involves DNA dep. RNA Pol II of the host
- "Vaccination" possible by avirulent form
82
Describe the viroids properties and give one example
Bsp.: Coco palms cadang-cadang viroid
- smallest infectious agents -> circular RNA
- covalently closed
- does not encode proteins
- are acellular particles
- like viruses; viroids also go through the cell to cell movement via plasmodesmata and systemic transport via the phloem
- the viroids replication cycle follows the same pathway of viruses, as they also function as the obligate intracellular parasites
- its replication requires RNA polymerase II to synthesize messenger RNA from DNA, by using viroid RNA as a template
- they can induce symptoms in higher plants via a mechanism known as "RNA silencing"
83
Genome replication of DNA viruses
- Genome amplification of SV40 involves the action of Topoisomerase and Primase.
- Amplification of Polyoma virus SV40 genomic DNA is similar to eukaryotic DNA replication.
84
Influenza Virus -> Ankreuzaufgabe
- Base pairing between both ends of each genome segment
- PA catalyzes cleavage of cellular pre-mRNA
- Uses cellular splicing apparatus
85
List four different methods for quantification of Influenza virus particles (with corresponding unit name). Explain why you get different numbers for the same virus preparation with these methods.
Number of viruses always depends on the method used for determination!
Infectious particles (in a sec. system!) vs total particles vs subunits!
PLAQUE ASSAY
Ability of a single infectious viral particle to form a macroscopical, cytopathic effect in a monolayer of cultivated cells!
- Isatin beta-Thiosemicarbazone (IBT): Vaccinia virus inhibitor
-> Quantifies only infectious particles = PFU (plaque forming units)
FOCUS ASSAY
Ability of a single infectious viral particle to form a macroscopical cytopathic effect in a monolayer of cultivated cells (here: Transformation of cells by a retrovirus)
DIRECT QUANTIFICATION BY EM
POCK ASSAY
- "Pock Formation"
- on the chorioallaintois membrane of chicken embryos
HEMAGLUTINATION (HA) ASSAY
Certain virions (e.g. Influenza) can bind directly (via their Hemaglutinin protein) to N-acetylneuraminic acid on the surface of erythrocytes. Due to the fact, that both, viruses and erythrocytes, have multiple binding sites, viruses can lead to the formation of cell latices (not visible). Without a virus erythrocytes settle out pf suspensions forming a "red dot". By starting with a defined number of erythrocytes viruses can be counted as "HA units".
-> HA units correlate with amount of viral hemagglutinin in a virus preparation.
-> Important values for vaccine production!
-> Variation there of: HA inhibition assay: By the addition of antibodies against the viral hemagglutinin the effect of the virus on erythrocytes can be inhibited: thus the amount of antibodies or virus can be calculated.
86
Many RNA Viruses have capped genomic RNAs similar to eukaryotic host mRNAs and use different mechanisms to produce 5' capped viral mRNAs. Place each virus family according to their mechanism used to generate capped genomic RNAs
1) De novo synthesis by cellular enzymes
2) Synthesis by viral enzymes
3) Acquisition of preformed 5' cap structures from cellular pre-mRNAs or mRNAs
DE NOVO SYNTHESIS BY CELLULAR ENZYMES
- Adenoviridae
- Hepadnaviridae
- Herpesviridae
- Papillomaviridae
SYNTHESIS BY VIRAL ENZYMES
- Poxviridae
- Reoviridae
- Rhabdoviridae
- Togaviridae
ACQUISITION OF PREFORMED 5'CAP STRUCTURES FROM CELLULAR PRE-MRNAS OR MRNAS
- Bunyaviridae
- Orthomyxoviridae
87
Animal viruses are completely dependent on the host cell machinery for translation of their mRNAs. Many of those viruses are using translation strategies to maximize the coding capacity their genomes of limited size. Name three principal strategies by which viruses achieve the maximization of the coding capacity of their genomes and state one viral example for each of them.
1) functional polycistronic mRNAs: Born disease virus (BDV)
2) read through of stop codons: Colorado tick fever virus
3) Polyproteins: Chikungunya Virus, West-Nile-Virus
88
Several viruses use IRES-elements for the initiation of translation of their viral proteins, which are classified into different types.
- Picornaviral IRES-elements (type 1 and type 2) do not require eIF4E.
- The turnip yellow mosaic virus (TYMV) contains a tRNA- like structural element in the 3'UTR that recruits ribosomes and places the transaction factors proximal to the 5' initiation site.
- Class IV IRESs of the Dicistroviridae, including CrPV, requiring only Eegs for polypeptide chain formation.
89
Rotaviruses use the viral nsp3 protein to interfere with host mRNA translation. Explain the underlying mechanism. Why can the translation of viral mRNAs still take place?
(Rotavirus: viral mRNA has cap, but no poly (A))
- viral NSP3 binds to eIF4G ands blocks the interaction eIF4G-PABP
- viral NSP3 binds to the 3' end of the viral mRNA and replaces PABP in stimulation of translation for viral mRNA
after binding of Rotavirus protein -> cellular mRNA is released
-> free complex binds viral mRNA
-> specific binding of viral mRNA to NSP3
-> Translation of viral mRNA
90
Briefly describe the course of Poliovirus minus-strand synthesis including a scheme depicting the critical genomic elements
- Product of minus-strand synthesis is dsRNA (RF, replicative form)
- multiple plus-strand synthesis occurs on one minus strand (RI, Replicative Intermediate)
1) Cloverleaf at 5' end of (+) genome binds 3CDpro and PCbp; PAbp binds PolA at 3' end of genome proximity of the ends; "circular"
2) VPN-peptide in 3AB (membrane protein) is cleaved off by 3CDpro
3) Binding of 3Dpol, 3CDpro and Veg to CRE
4) AAACA sequence in CRE is a template for VPg uridylylation, VPgpUpu
5) Transfer of VPgpUpU to the 3' end of the genome as a complex made of VPgpUpU, 3Dpol and 3CDpro
6) Annealing of pUpU to polyA tail
7) Elongation of (-) strand
91
List the 4 types of persistent infections and give one viral example each (species and family).
1) suppression of the innate immune system/apoptosis -> Flaviviridae: HCV
2) very high antigenic variability -> Retrovirus: HIV and Flaviviridae: HCV
3) intrauterine infection -> Flaviviridae: BVDV
4) suppression of the innate immune system -> Paramyxoviridae: measles virus and Herpesviridae: EBV
92
Viruses causing hepatitis
HEPATITIS A VIRUS
family: Picornaviridae
Genome: RNA 7,5 kb
Persistent infection: no
Fecal oral infection: yes
HEPATITIS B VIRUS
family: Hepadnaviridae
Genome: DNA 3200 bp
Persistent infection: (yes)
Fecal oral infection: no
HEPATITIS C VIRUS
family: Flaviviridae
Genome: RNA 9,5 kb
Fecal oral infection: no - blood
HEPATITIS E VIRUS
family: Hepeviridae
Genome: RNA 7,2 kb
Fecal oral infection: yes
93
Why was HBV first described as Australia antigen? (one sentence)
Das Australia-Antigen wurde als erstes im Blut von australischen Ureinwohnern gefunden.
94
What are Dane particles and what do they consist of?
("virusähnliche Partikel")
- Durchmesser von 42 nm
- Überschüssig produziertes Virusoberflächenantigen
- particles in the blood of patients with "serum hepatitis"
95
Explain the term and mechanism "attenuation of transcription" for negative strand RNA viruses.
- start of mRNA synthesis always at 3' end of N gene (not 3' end of genome)
- inefficient reinitiaiton after termination of transcription
-> Termination of mRNA synthesis at "intergenic region" (ig)
ATTENUATION: decreasing amounts of transcript from 3' to 5' (N>P>M>G>L) leading to severely reduced amounts of L (RdRp) (but high levels of N)
- PolyA via stuttering of viral polymerase at the 7xU sequence in the "intergenic region"
96
Name one virus family and one virus which uses the "attenuation of transcription" mechanism. Draw schematically the genome organization of a typical representative of this family.
- Vesicular stomatitis virus (VSV) -> family: Rhabdoviridae
3' Leader ig N ig P ig M
97
To which family does the Dengue Virus belong?
Flaviviridae
98
Why is the Dengue Virus classified as ARBO virus and what does this abbreviation stand for?
- durch Anthropoden übertragbare Viren (Gliederfüßler wie Moskitos, Sandfliegen und Zecken)
99
Why is it more complex to develop a Dengue vaccine compared to yellow fever? Explain briefly the critical aspects.
- pathomechanism of severe dengue is still poorly understood -> most plausible hypothesis: antibody dependent enhancement in secondary infection
- lack of an appropriate animal model
- poor knowledge of correlates -> both for protection and disease enhancement
- interaction among the four serotypes -> as a tetravalent immune response is desired, when a mixture of all four serotypes in a tetravalent live attenuated vaccine is given, each component would need to independently result in four different monotypic immune responses that are solid to each serotype
100
What are the actual approaches to solve the problems of the development of a Dengue vaccine?
CYD-TDV: tetravalent live attenuated dengue vaccine with a yellow fever 17 D backbone -> contains mixture of four dengue serotypes designed to induce long-lasting neutralizing antibodies
-> this leads to competition among serotypes with respect to replication and the ability to stimulate neutralizing antibodies, probably owing to the activation of toll-like receptors and the induction of innate immunity -> therefore complete immunization may require multiple doses
- interference manifested by asymmetric immunological responses to the mixtures of you dengue vaccine viruses
101
Explain the basic mechanism of the initiation of genome replication of Dengue virus including a basic scheme for illustration.
1) Cyclisation of the genome via base pairing
2) Binding of RdRp to 5' end
3) Transfer of polymerase from 5' to 3' end
4) Start of RNA synthesis at 3' end
- no initiation at 3' UTR fragment!
- annealing of RNS genome ends allows RdRp to initiate RNA synthesis at both 3' ends (two products)
- replication initiation in trans: binding of RdRp to 5' UTR allows initiation at 3' end
- Deletion of poly U stretch upstream of 5' UAR eliminates initiation at 3' end of 3' genome end
- spacer required for flexibility
102
(+) strand RNA viruses restructure the intracellular membrane systems of the host cell for their genome replication. Name for each of the compartments listed blow one virus which makes use of these membranes for genome replication.
ER: Polioviurs, HCV
Golgi: Alphavirus
Lysosomen: Alphavirus -> Simian foamy virus
Mitochondrien: Flock house virus
103
By which model were 3D models of the virus-induced membrane structures obtained?
Electron tomography (ET) -> 200-400 nm thick sections at 3-8 nm resolution are recorded from different angles to obtain a stack of 80-140 images
104
Describe the pathway used by SV40 to its site of genome replication.
DNA replication highly similar to eukaryotic cell!
1) T-Antigen (T-Ag) binds as 2 hexameters in the presence of ATP; strand melting of A/T rich regions
2) In the presence of the heterotrimeric "replication protein A" (RPA; binds ssDNA) unwinding of DNA occurs (T-Ag helices) under hydrolysis of ATP
3) DNA Pol alpha/primase binds to each single strand and synthesizes short pieces of RNA serving as primers for DNA synthesis. In this process the interaction between Pol alpha/Primase and T-Ag (as chaperon) is important. Pol alpha/Primase are then replaced by replication factor c and "Pol delta complex".
105
At which intracellular membrane system does the RNA replication of the following viruses occur?
SARS Coronavirus
Sindhis virus (Alphavirus)
Flockhouse virus
Poliovirus
SARS Coronavirus: ER
Sindhis virus (Alphavirus): lysosomes
Flockhouse virus: Mitochondria
Poliovirus: ER
106
RIG-I is an important protein in the innate immune response
-> Describe the properties of RIG I
- retinoid acid inducible gene I
- Helikase -> intrazellular receptor
- restriction factor
- recognizes RNA viruses like Hepatitis C, Influenza
- pattern recognition receptor
107
Describe the signaling cascade of RIG I and name the relevant elements
1) In the absence of viral RNA, RIG-I patrols the cell with the CARDs packed tightly against the other protein domains
2) Upon viral entry and generation of a blunt dsRNA with two or three phosphates (p3dsRNA)
3) RIG-I binds tightly to the p3dsRNA terminus, and the tethered CARDs are displaced into the cytosol
4) Liberated CARDs are then modified by cofactors that append ubiquitin, which prevents return to auto pressed state
5) Finally, active RIG-I moves to the mitochondria and nucleates MAVS oligomerization
108
At which step does Influenza virus interfere with the RIG I signaling pathway? Which viral protein is required for interference.
Ubiquitination of the RIG-I2CARDs by the E3-ubiquitin ligase TRIM25 is a critical step in activation of the RIG-I signaling pathway. This step is inhibited during influenza infection by a direct interaction between NS1 and TRIM25.
109
The cellular miR-122 is a critical host factor for the Hepatitis C Virus life cycle. Which of the statements concerning the mechanistic and/or functional features for miR-122's role in the HCV life cycle given below are correct?
- The cellular miR-122 binds to the 3'UTR and to the 5'UTR of the viral genome.
- miR-122 inhibits HCV RNA synthesis by displacing PCBP2 from the viral RNA genome. The microRNA miR-122 competes with PCBP for binding to the 3'UTR.
- miR-122 supports HCV RNA stability and HCV RNA replication by protecting the 5' end from Vrn1-mediated degradation.
110
Viruses take active counter measures against the interferon induction via the RIG-I pathway. Give one example of a (+)-strand and one example of a (-)-strand RNA virus and explain which viral proteins are used to antagonize their recognition by this pathway. Name the viral antagonists and their mode of action.
(-)-strand RNA virus: Influenza virus
-> Ubiquitination of the RIG-I2 CARDs by the E3 ubiquitin ligase TRIM25 is a critical step in activation of the RIG-I signaling pathway. This step is inhibited by a direct interaction between NS1 and TRIM25.
(+)-strand RNA virus: SARS CoV2
-> membrane protein, encoded by ORF5 functions as an IFN antagonist via direct interactions with RLRs and MAVS to prevent RLR/MAVS/TRAF3/TBK1-containing signal complex formation.
111
A minority of viruses utilize non-canonical mechanisms in the biogenesis of miRNA molecules. Which of the following answers is/are correct?
- some viral pre-miRNAs are transcribed by RNA polymerase III
- all viral pre-miRNAs are exported into the cytoplasm by exportin-5
- all viral miRNA precursors are processed by Drosha
- all viral pre-mRNAs are processed by dicer
112
Ankreuzaufgaben zur (-)-Synthese von RNA-Viren
- Visionen von (-)-Strand RNA Viren beinhalten virale RdRp
- RNA Replikation von (-)-Strang RNA Viren findet immer im Cytoplasma statt
113
- Picornaviren besitzen eine Hülle
- Picornaviren Protease spaltet nur virale Proteine
- Produkt der (-)-Strang Synthese wird "Replikatives Intermediat" genannt
- Das Picornavirus Genom hat einen Poly-A-Schwanz am 3' Ende
114
Welche der Viren nutzt den zellulären Splicing Apparat?
Influnezavirus
115
Influenza Virus: Kreuzen Sie korrekte Antworten an!
- Pa katalysiert die Spaltung der zellulären pre-mRNA
- Genomsegmente haben eine geordnete Struktur im Virion
116
Mehrere Viren nutzen IRES-Elemente für die Initiation der Translation ihrer viralen Proteine, welche in verschiedenen Typen klassifiziert sind. -> Ankreuzfrage
- Picornavirale IRES-Elemente (Typ 1 und Typ 2) benötigen kein EIF4E
- HCV: Transaltionsinitiation benötigt ein "scanning" der ribosomalen 40S-Untereinheit vom 5' Ende der viralen mRNA
- Translationsinitiation von der HCV IRES ist abhängig von eIF2
117
Sendai Viren nutzen verschiedene Strategien für die Expression von Genprodukten von den P/C Genen. Kreuzen Sie korrekte Antworten an!
- Expression von Protein C', P und C tritt nicht im gleichen reading frame auf
- Translation von Protein P, W und V beginnt am gleichen Startcodon.
- RNA editing wird benötigt für die Expression von Protein W und V.
118
Durch welchen Mechanismus schaffen Coronaviren eine differentielle Regulation der Menge von Replikase Proteinen? Erklären Sie kurz den Mechanismus und die Elemente, welche an diesem Prozess beteiligt sind.
ANTIHOST FUNCTION OF COV NSP1
- binds to 40S ribosomal subunit
- stops translation of cellular mRNA (non-specific, includes IFN mRNAs)
- modifies 5' end of mRNA to render it translationally incompetent
- later discovered: induces endonucleolytic cleavage of mRNA near to 5' end -> nsp1 is most likely NO nuclease itself -> host nuclease
- leader sequence in SARS coronavirus mRNAs protects against mediated degradation
119
Rotaviren nutzen das virale nsp3 Protein, um mit der Wirtszell mRNA Translation zu interferieren. Beschreiben Sie den Mechanismus. Wieso kann die Translation viraler mRNA trotzdem stattfinden?
-> Dimer binds 3' end of viral mRNAs, competes with PABP for eIF4G-1 binding, so prevents cyclization and translation of cellular mRNAs
120
HBV: Kreuzen Sie die korrekten Antworten an!
- causes liver damage by immune cells not by viral cytopathogenicity
- mRNAs is transcribed by viral polymerase
- reverse transcription takes place in immature nucleocapsid
- genome replication includes 3 template switches
- RNA contains a packaging signal
121
Kreuzen Sie die richtigen Aussagen über die Blauzungenkrankheit an
- Blauzungenkrankheit ist resistent gegen Detergenzien
- Die Blauzungemkrankheit besitzt Glykoproteine
122
Beschreibe den Baltimore Scheme
Group I: dsDNA -> Beispiel Adenoviridae
Group II: ssDNA -> Beispiel: Parvoviridae
Group III: dsRNA -> Beispiel,: Reoviridae
Group IV: (+) ssRNA -> Beispiel: Flaviviridae (-> (-) ssRNA)
Group V: (-) ssRNA -> Beispiel: Orthomyxoviridae
Group VI: (-) ssRNA -> Beispiel: Retroviridae (-> (-) ssDNA)
Group VII: partielle dsDNA -> Beispiel: Hepadnaviridae
123
Immunological reactions against virus infections
1) Innate immune system
- interferon system
- macrophages
- "natural" killer cells
2) Cellular immune response
- T-killer cells (CD 8)
- T-helper cells (CD4)
3) Antibodies
- neutralizing Abs are important for the protection against reinfection
- main determinant for success in vaccination
- does not always protect against infection with identical pathogens e.g. influenza virus, hepatitis C virus, Hi virus
124
State one viral example (virus and family) for each of the possible polarities
(+) ssRNA: Flaviviridae -> Hepatitis C
(-) ssRNA Orthomyxoviridae -> Influenza A Virus oder Hepadna viridae -> Hepatitis Delta Virus
125
Flavivirus: division into two major groups
-> based on the vector they utilize for transmission
-> mosquito borne flavivruses (bsp. Yellow Fever virus, Dengue virus)
-> tick-borne encephalitis
-> not vector borne -> yet unidentified arthropod vector
126
Beschreibung IRES Strukturen
- spezifische Faltung (Sekundärstruktur) der mRNA
- vermittelt ohne Initiationsfaktoren die Bindung der mRNA an die Ribosomen
- ermöglicht bei der >Synthese von Proteinen in Eukaryonten die Translation unabhängig von der 5' Struktur zu initiieren (bsp. auch von der Mitte der mRNA aus)
- kommt in der RNA von Viren und mRNA einiger Gene von Zellen vor
- Bsp. HCV, Poliovirus
127
Glycoprotein G of Vesicular Stomatitis Virus
- involved in receptor recognition at the host cell surface
- after endocytosis of the vision -> triggers membrane fusion via a low pH-induced structural rearrangement
- atypical fusion protein: pH-dependent equilibrium between pre- and post fusion conformations
- homotrimeres Andockprotein
- nach der Bindung des VSV-G an die Zelloberfläche erfolgt die Cathrin-vermittelte Endozytose des Virions in ein Endosom
- wird zur Pseudotypisierung von viralen Vektoren verwendet
128
Difference between the polarities
(+) STRAND RNA VIRUS
- can directly be translated into viral proteins by host cell ribosomes
- encode an RdRp for replication
(-) STRAND RNA VIRUS
- viral RdRp produces mRNA -> during replication a (+) antigenome is produced -> template to create genomic (-) RNA
- contain viral envelope that surrounds capsid
- are usually linear and commonly segmented
129
Influenza A Virus can block the induction interferon alpha/beta with one of its gene products
NS1: can inhibit interferon beta
-> blocks activation of IFN regulatory factor 3 (IRF3) and blocking post transcriptional processing of cellular mRNAs
