VL 1 Introduction
Able to recognize viral replication cycles of some important virus families (adenoviruses, hepadnaviruses, herpesviruses, orthomyxoviruses, picornaviruses, retroviruses, nidoviruses)
Are able to name important characteristics within the replication cycles of these viruses (e.g. RNA/DNA genome, mechanism of entry, site of replication (nucleus/cytoplasm), mechanism of release, etc.
VL1
Zusatz? (not listed in questions)
Poliovirus
Attachment and Entry
Release of Genome (Uncoating)
Translation of viral proteins
Genome replication
Assembly
Release
(sometimes: maturation)
Hepadnaviruses
enveloped DNA virus
ccc DNA
replication in nucleus
progenmoic RNA
reverse transcription in virion (RNA→ DNA)
release by nonlytic exocytosis
Adenovirus
non-enveloped DNA virus
entry via endocytosis
immediate early (E1A cell cylce protein →S phase), early (viral polymerase) and late (structural proteins) gene expression
assembly in nucleus
release by lysis
Herpesviruses
orthomyxoviruses
picornaviruses
retroviruses
Nidoviruses
Recognize or sketch the different compounds of enveloped an non-enveloped viral particles
1.Draw an enveloped virus
2.Draw an non enveloped virus
know what syncytium formation is and its mechanism
enveloped viruses enter their host cell by membrane fusion
fusion of viral envelope and cell membrane is triggered by the envelope proteins
after cells are infected with enveloped viruses, they express viral proteins on their surface
close contact to uninfected cells can lead to cell/cell membrane fusion and the formation of syncytia
Syncytium formation is a classical CPE of enveloped viruses.
= Cytopathic Effect
Can determine a CCID50 from a graph
/ How can you determine the ccID50 from a graph showing infected cells depending on virus concentration
(The Endpoint-Dilution Assay)
Cell culture infectious dose concentration of virus at which point 50% of the cells are infected
know the difference between physical and biological virus titer and know methods how to determine these titers
General: The virus titer is the concentration of a virus in a suspension.
infectious titer/biologial titer
concenration of infectious particles in a suspension (depends on test system, e.g. celltype
test via Plaque assay, transformation assay or flow cyometry
physical titer
concentration of infectious and noninfectious particles
meassure by ELISA or TaqMan-PCR
know basic methods used in virological labs (cell culture, 3D-cell culture, organoid culture, fluorescent viruses, immunohistochemistry, TaqMqn-PCR, plaque assay)
vgl. verschiedene Karten
cell culture
What is the difference between 2D, 3D cell culture and organoids
2D flat cells
3D tissue rebuilt with cells in 3D and scaffold
organoid: minimized version of an organ produced in vitro in three dimensions by IPSCs or ESCs
3D-Cell culture
Organoid Culture
A 3D cell culture is an artificially created environment in which biological cells are permitted to grow or interact with their surroundings in all three dimensions
Describe fluorescent viruses and immunhistochemistry
Fluorescent viruses:
transgenic viruses that express fluorescent proteins
immunhistochemistry
use fluorescent labeled anti virus antibodies
Plaque Assay
dilute virus stock solution to different concentrations
distribute virus on cell lawn
count PFus at different C and reestimate origional concentration in stock
__________________
Virus suspension z.B Log verdünnen dann Zellkulturen infizieren dann kann man es anhandvon Plaques erkennen
Klausur siehe Glossar: Pfui plaque forming unit —> Menge mit so einem Assay bestimmt
TaqMan-PCR
specific technique for amplification and simultaneous quantification of DNA content
specific flurorescence signal by dual-labeled probes ("reporter" and "quencher").
the reporter is released by 5´-exonuclease activity of the Taq polymerase (hydrolysis of the probe); destroying the conncection to the quencher
Release of the reporter leads to fluorescence of the reporter (light is no longer absorbed by the quencher)
High specificity (PCR only provides signal if both primers and probe match)
Standard procedure to quantify viral DNA
(working with RNA-Viruses requires reverse transcription of the RNA genome into DNA prior to PCR)
know what a receptor, coreceptor or attachment factor is
Attachment factor or adhesion receptor are common extracellular molecules that make the first contact to a virus which increase the probability of the virus to stay at the cell surface
They increase the probability that the virus stays at the cell surface e.g. heparane sulfate or silaic acid
Receptor: mediates viral entry into the cell, determines tropism of virus (the virus can only enter cells that express this molecule), influence host range e.g. CD4
Coreceptor: second binding receptor, needed for viral entry e.g. CCR5
know the definition of the Kd for receptor/ligand complexes
Dissociation constant, that describes the binding strenght of a ligand/receptor-binding
can describe the entry of HIV and influenza in detail
(+ measels)
HIV:
Binding of gp120 to CD4/CCR5 triggers a conformation change in gp120 that enables the attack of gp41 to the host membrane
Influenza:
A drop in pH in the endosome triggers a conformation change in measles H protein that enables the attack of measles fusion protein to the host membrane
Measles:
after receptor binding, the viral H protein undergoes a conformational change allowing the F protein to attack the host membrane
can name examples for viral entry of enveloped and nonenveloped viruses and can name principal differences
What is the difference of virus entry by HIV, measles, influenza and adenoviruses? Which of these viruses are enveloped`
Enveloped (taken up by membrane fusion: directly at plasma membrane or in endosome)
HIV/measles: fusion with cell membrane
influenza: uptake via endocytosis; fusion with endosomal membrane
Non-enveloped (always taken up by endocytosis)
Adenovirusues: uptake by endocytosis and disruption of the endosomal membrane (via protein VI)
are familiar with schematic representations of receptor-binding surface proteins and their domains VL1
Non-enveloped virus: Surface structures (grooves and loops) and fibers like spikes
Enveloped virus: Transmembrane glycoproteins: mushroom with receptor binding domain (klappt weg) and fusion domain (nun chuck domain)
Zusatz
How are Lysosomes derived and name properties
derived from late endosome after further pH decrease
contain proteases, nucleases and lipase
have a pH of 4.5 to 5
Baltimore Classification
How to get to mRNA from the genome
All viruses must produce mRNA that can be translated by cellular ribosomes. In his classification system, the unique pathways from various viral genomes to mRNA define specific virus classes on the basis of the nature and polarity o their genome.
Virus classification according to the viral genome
VL2 Hepatitis Viruses
What 5 viruses can cause hepatitis?
VL2
What viruses can cause hepatitis?
-Hepatitis A-D (HAV, HBV, HCV, HDV)
-ABV
-CMV
-HSV1
-VZV
-Coxsackie-Viruses
-Yellow Fever Virus
Which hepatitis viruses can cause chronic disease?
Hepatitis B, C, D (HBV, HCV, HDV)
HBV: 5-10% adults, 90% newborns
HCV: 50-80%
HDV: Co- 5-10, super 70-95%
Hepatitis A: never!
Hepatitis E: rare (in immunocomprised)
What is the route of transmission?
unprotected sex (65%)
i. v. drug abuse (needle sharing)
parental (blood)
sporadic in ca. 30% Tattoos, Piercings, Acupuncture, smear infection
perinatal
________________________Valentin
HAV: fecal-oral
HBV: sex or parenteral or iv drugs
HCV: sex or parenteral or iv drugs
HDV: parenteral or iv drugs
HEV: fecal oral or zoonotic
Which hepatitis virus infections can be prevented by vaccination?
Hepatitis A (Active/passive)
Hepatitis B (Avtive(passive)
Hepatitis C (no)
Hepatitis D (via HBV)
Hepatitis E (no)
Replication cycle of HBV
virus is encapsulated at the surface
capsid migrates to nucleus and releases partial dsDNA into nucleus
completion of partial dsDNA into dsDNA after entry in the nucleus
formation of cccDNA with histones
cccDNA serves as template for +ssRNA and multiple mRNAs
translation of viral proteins from the mRNAs
reverse transcription of +ssRNA pregenome starts in the assembled particle
dsDNA synthesis stops when all nucleotides in the particle are used up
release by exocytosis
How does the serology of hepatitis with healing differ from the chronic one?
VL 2 Zusatz
In chronic no anti HBs production
Comparison early and late reverse transcription of HBV and retroviruses. Is reverse transcription important for the immunce escape?
in retroviruses the reverse transcription from RNA to DNA takes place early in the replication cycle, before RNA enters the nucleus
in hepadnaviruses like HBV reverse transcription takes place late when the virus particle is assembled
the RT of HIV is important for immune response escape
HBV RT also has a similar bad copying mechanism, however mutations are usually lethal as the genome is rather small and overlapping
How can HBV infection be treated?
Valentin
with reverse transcription inhibitors like Tenofovir
it is a nucleotide analogon which lacks the 3’ OH causing chain termination during HBV rplication
lifelong therapy because cccDNA is stable in the liver and the treatment does not lead to anti HBS production
with vaccination
with recombinant HBs antigen
recommended for infants since 1995
_____________________________Kim
--> no elimination
—> HBV therapy is life-long. It can protect the liver but it cannot eliminate the virus from the body.
cccDNA is very stable and last lifelong in the liver
The therapy does not trigger anti-HBS antibodies: no immunity is being formed to control HBV replication when therapy is being stopped.
Hepatitis-B-Therapy
Reduction of viral load, preservation of the liver, but no cure.
Substances: Reverse transcriptase inhibitors
Vaccine:
Vaccination with recombinant HBs antigen (inactivated vaccination)
For infants: Recommended since 1995 as part of basic immunization
Recommended for at-risk groups, incl. health care providers
For adults: Triple vaccination (0 - 1 - 6 months)
For health care workers and other risk groups: HBs antibody control 1 - 2 months after last vaccination.
HBs-Ak < 100 IU/l: immediate booster vaccination.
HBs-Ak ≥ 100 IU/l: titer control after 10 years, booster vaccination if < 100 IU
Replication cycle of HCV
entry by receptor mediated endocytosis
fusion with endosome membrane at low pH
direct translation of +ssRNA genome at ribosomal entry site
IRES sequences used for initiation of translation
genome replication with RNA dependend RNA polymerase uses -ssRNA as a template
protelytic cleavage of precursor protein by viral proteases
assembly into ER
release by golgi apparatus by secretory pathway
Compare the HCV genome with normal mRNAS/
Genome of HCV: similarities and differences with mRNAs
mRNA has a cap structure, HCV has an IRES site
mRNA is monocistronic, HCV-RNA is also monocistronic but resulting in a large precurser protein that is cleaved into smaller proteins by proteases
mRNA has a poly A tail, HCV RNA has no poly A tail but a 3’ UTR
normal mRNAs
5’ cap
5’ UTR
start codon (AUG)
ORF
stop codon (UAA, UGA, UAG)
3’ UTR
PolyAtail
usually monocystronic (only contains info for one protein)
translation initiation is cap dependent
ssRNA genome of HCV
no cap but IRES after 5`UTR
polycistronic RNA → individual proteins produced by proteolytic cleavage of large precurser protein
What is an IRES and what is it used for in HCV replication? In molecular biology?
An IRES (internal ribosomal entry site)
structure within an mRNA molecule that allows the binding of the 40S subunit in the absence of a cap structure
IRES in molecular biology
used to create polycistronic mRNAs
frequently used to coexpress a reporter-gene or resistance-gene together with a gene of interest with only one promotor needed
What is an IRES and what is it used for in and molecular biology
Zusatz VL2
IRES sequenced used for initiation of translation
How can HCV infection be treated?
Combination of 2-3 of the following substance classes is used.
Protease inhibitor (…previr)
Polymerase inhibitora (…buvir)
NS5A-Inhibitors (…asvir) (NS5A regulates replication and virus assembly
Ribavirin (change of the equilibrium of the intracellular nucleotide concentration)
Therapy takes 8-12 weeks, then the patient is cured. Due to lack of any persistence mechanism, HCV is completely eradicated from the body after therapy
Why can HCV be eliminated by therapy, but HBV infection cannot?
HCV is a pure mRNA virus and doesnt enter the nucleus, therefore it does not have a persistance mechanism like the formation of cccDNA in HBV
What gene does the HBV genome encode for?
RT
X
surface
core
VL3 HIV
What is a retrovirus? What genus does HIV belong to?
VL3
viruses that have a RNA genome
reverse transcribe their genome into DNA
the DNA genome is integrated into the cellular genome → necessary step of replication
HIV belongs to the genus Lentiviruses
Lentiviruses belong to the subfamily of orthoretroviruses containing alpha, beta, gamma, delta and epsilon retroviruses besides Lentiviruses
What is an endogenous retrovirus?
inherited proviral sequence
endogenous viral element in the genome that closely resemble and can be derived from retroviruses
example for a retroelement
What are retroelements?
self-serving DNA (eigennützig) 42% of human genome
jumping elements in the genome which have a RNA intermediate
sit at characteristic locations in the DNA strand
coded by an intern and therefore mitwanderndem promotor
trancription to RNA and then rewriting to DNA guarantees spreading
elements in the human genome that shows retroviral pattern
e.g. HERVs → human endogenous retro viruses → element with gag, pol, env between two LTRs
What are PBS, LTR and ppt?
PBS → primer binding site
LTR → long terminal repeats ( U3 R and U5)
ppt → polyppurine tract
What is a Provirus?
VL4
the DNA genome integrated into th ehost genome
additional U3 and U5 region is added when compared to the sequence in the particle
Sketch the retroviral replication cycle
entry
uncoating
reverse transcription
integration in genome →provirus
expresion
assembly
release
Know the 3 enzymatic activities of reverse transcriptase
RNA-dependent DNA polymerase activity (synthesis of 1st DNA strand)
DNA-dependent DNA polymerase activity (synthesis of 2nd DNA strand)
Rnase H (heteroduplex exonuclease) activity (degradation of RNA strand)
Know the essential elements of reverse transcription in vivo/in vitro
KLAUSUR
Know the RT process of HIV in “detail”
Rt uses tRNA lys as primer for first DNA strand. tRNA is bound to PBS near the 5’ end of the viral genome → synthesis of short DNA strand and removal of RNA strand
identical R regions at both ends of the retroviral genome allow base pairing of the old strand and complementary new strand→ first jump of the RT via pseudocirculation -synthesis of new strand
RNaseH activity in RT degrades RNA template
ppt region is resistant to RNaseH and serves as a primer for the second DNA-strand
the PBS from the first DNA transcript and the newly produced complementary PBS from the second DNA transcript also allow base pairing → second jump of the RT via pseudocirculation
Can compare early and late reverse transcription of HIV/HBV
in HIV: early reverse transcription to DNA, before integration into the host genome // RT early (+) ssRNA genome into cDNA
in HBV late reverse transcription inside of the assembled virus particle// RT late ssRNA genome into dsDNA
Know the difference between productive and latent infection and reactivation
Entry, reverse transcription, integration, expression and assembly, release, maturation
productive infection: 1 in 100 T cells: integration into genome and expression of viral genes
latent infection: 1 in 1 mio cells: not recognized by the immune system → no viral proteins produced
reactivation: expression of proteins from provirus
Are familiar with HIV origin
Origin:
transmitted from monkeys to humans approx. 1880-1920
Origin: Central Africa, Congo
There are 2 HIV types
Type 1: Pandemic type, Origin: Chimpanzee
Type 2: Endemic in West Africa, Origin: Sooty Mangabey
There are several subtypes of HIV-1 (e.g. A, B, C and recombinant).
Main subtypes in EU/USA: subtype B
Main subtypes in Africa: subtype C
HIV therapy works equally well for HIV-1 and HIV-2 and for the different HIV-1 subtypes.
What is the pathigenesis of HIV? what are the reasons for this phenomenon?
Pathogenesis:
Depletion of CD4 cells in the intestine (acute phase)
Continuous loss of CD4 cells due to immune activation/depletion anti-HIV CTL apoptosis
High viral load correlates with rapid progression
High immune activation correlates with rapid progression
Rare: long-term nonprogression (control of infection for decades)
Name HIV therapy options
Valentin:
Fusion inhibitors
receptor antagonists
Reverse Transcriptase Inhibitors (NRTIs, NNRTs)
Integrase inhibitors
Protease inhibitors
⇒ combinational therapy of 2 nucleotide RT inhibitors (NTRIs) and one non nucleotide RT inhibitor (NNRTI), a Protease inhibitor or an integrase inhibitor directly after positive diagnosis
before start of therapy genotypic resistance test
PCR to monitor viral load during therapy
therapy goal: reduction of viral load below detection limit
no end of therapy
therapy improves immune system and prevents AIDS and HAND → normal life expectation
Therapy:
Therapy is usually being started with one of these combinations
A combination of 3 NRTI is inferior compared to the above-described combinations.
HIV therapy is always a combination therapy
2 NRTI +1 NNRTI
2 NRTI +1 PI
2 NRTI +1 INI
Start of therapy immediately after positive diagnosis
Before therapy start: genotypic resistance test
Therapy goal: reduction of viral load below detection limit
Duration of therapy: unlimited, no cure possible
Therapy improves immune system (CD4-count in blood increases within months to years to normal values: approx. 1000/ml, CD4-count in intestinal flora increases much slower), prevents AIDS, prevents HAND
Life expectancy under therapy: normal
What to do after exposure to HIV?
VL3 ZUSATZ
enhance bleeding
alcoholic wound cleaning
initiation of Post exposure prophylaxis within 2hours
HAART (highly active anti retroviral therpy for 4 weeks
HIV testing at day 0 and month 1,3,6 post HAART
Why is there no HIV vaccine?
Zusatz VL3
HIV quasispecies with high antigenic diversity
there is no immunity with HIV after infection
superinfections possible even after long time
What is the tropism of HIV
receptor CD4
coreceptor CCR5 or CXCR4
tropism:
CD4 pos T cells
macrophages
dentritic cells
microglia
⇒ mainly production in activated T helper cells
What genes does HIV express?
VL3 Zusatz
retrovirus genes:
gag: encodes capsid and matrix
pol: encodes PR, RT and IN
env: encodes envelope proteins
accessory genes
tat, nef → regulatory proteins
Name properties of HIV clinic and what are results of HIV without proper treatment?
transmission: sexual, parenterally, during birth, breast milk
incubation period: 1-6 weeks
acute phase with general symptoms
symptom free phase for 2-12 years with increased T helper cell loss
without treatment AIDS (100%) and HAND (30%)
VL4 Herpesviruses
What is viral persistence?
virus remains in body over a long time or lifelong
What is viral latency? On what level is it controlled?
the virus persists but does not replicate
latency controlled on genome level e.g. circular viral DNA of Herpesvirues or integrated DNA of HIV
What is clinical latency?
the absence of symptoms→ not to be confused with the virological latency → e.g. HIV 2-10 years of clinical latency but with productive infection
Define productive infection
active viral replication
viral particles are formed
Define reactivation
switch from latency back to productive infection
Name 4 different types of infection patterns with viral examples
acute infection
influenza, rotavirus, SARS Cov2
Chronic infection (ongoing replication with some symptomatic phases
HBV, HCV
latent infection (virus never really leaves body, virus sleeps and awakes)
herpesviruses
slow infection
HIV
Name the HSV subfamilies and their location of persistence
HHV1-8
alpha subfamiliy - persistence in neuroganglia (non neural cells in CNS)
HHV1 (HSV1)
HHV2 (HSV2)
HHV3 (VZV)
beta subfamily - persistance in lymphocytes/ monocytes in the kideney and salivary glands
HHV5 (CMV)
HHV6
HHV7
gamma subfamily- persistence in B lymphocytes
HHV4 (EBV)
HHV8
Disease symptoms caused by HSV-1
(Zusatz HSV-2 and VZV)
HSV-1: cold sores
primary infection: stomatitis aphtosa (inflammation mundschleimhaut)
reactivation: mainly herpes labialis but also herpes genitalis
______________________________Zusatz:
HSV-2: genital ulcer
primary infection: vulvovaginitis herpetica (vaginalentzündung)
reactivation: mainly herpes genitalis but also labialis
VZV: chicken pox or varizell zooster (windpocken, Gürtelrose
Can describe the replication cycle of HSV-1: entry, concept of IE and E and late, genome replication, packaging, egress
attachment to heparane sulfate
binding to HVEM (herpes virus entry mediator) receptor (TNFa superfamily)
membrane fusion mediated by envelope
capsid transport to nucleus
release of HSV-DNA into nucleus via nuclear pore
Concept of IE and E and late:
HSV-1 packaging:
The concatemeric genome is pushed by the terminase protein into an empty capsid
Terminase also cleaves the concatemeric pregenome once a full copy of HSV genome is packed into the capsid
(packaging can be inhibited for the herpesvirus CMV using the drug Letermovir that is being used as CMV prophylaxis after stem cell transplantation
Describe the replication cycle of HSV-1
after entry, transcription in 3 phases (IE, E and L)
regulation of different phases controlled by viral regulatory proteins (VP16, ICP4, ICP0, ICP8)
DNA replication only in late phase
all phases several hours apart
entry, uncoating trancription, translation → after each step, the proteins that were translated migrate back to the nucleus
circularization of the linear genome
in late phase replication by theta replication and rolling circle replication
concatemeric DNA (consisting of repeating units) is cleaved into monomeric, linear DNA upon packaging
budding at golgi membrane
endosomal release
Theta replication
HSV has 3 redundant ORIs
theta replication involves two replication forks after replication has started on OriS
OriS binding protein binds → ICP8 ss binder binds → helicase-primase complex binds → DNA pol and processivity factor bind
intermediate structure looks like theta, results in 2 copies of circular DNA
rolling circle replication
main form of genome replication
only one replication fork that produces continously new DNA from the circular template
results in concatemeric DNA that consists of multiple HSV genomes → like rolling out a toilet paper
How does HSV-1 packaging take place?
concatemeric genome is pushed by the terminase into an empty capsid
terminase cleaves genome once one full progenome is packed into one capsid
... know the moa (mechanism of action) of aciclovir
nucleoside with missing 3’ OH group for replication
serves as prodrug
if cell is infected with alpha herpesvirus, the viral thymidine kinase can phosphorylate acyclovir → the resulting nucleotide analogon is incorporated into the viral genome by the viral DNA polymerase → strand breakage; replciation interrupted
Can describe the mechanism of latency and reactivation: retrograde transport, anterograde transport, LATs; please also learn miRNA and its effect on ICP4 and ICP0
Latency and reactivation
epithelial cells support productive infection
nerve terminals are infected
axonal (retrograde) transport of the capsid into the cell body in the ganglia
here viral genome delivered to nucleus
viral episome is stably maintained in the neural nucleus
latent infection established
upon reactivation new particles produced and transported by (anterograde) transport back to the epithelium
During latency, 4 latency-associated transcripts (LAT) are transcribed
They are processed into miRNAs that suppress the transactivators ICP4 and ICP0 that normally would activate the transition from IE to E phase.
What are LATs
latency associated transcripts
during latency 4 LATs are transcribed
they are processed into miRNAs which suppress transactivators ICP0 and ICP4 that normally initiate transition from IE to E phase
Which virus can cause mononucleosis (Pfeiffersches Drüsenfieber) and what are the symptoms of this disease?
VL4 Zusatz
EBV → name mononucleosis derived from high number of lymphocytes in the blood during EBV infection
similar symptoms as with HIV
swollen lymph nodes
pharyngitis (Entzündung des Rachens)
fever
feeling tired
Rash (Hautauschlag) when treated with antibiotics
EBV infection linked to multiple sclerosis
Know that EBV infection can cause infectious mononucleosis and its symptoms
EBV-Infections can be asymptomatic. However, especially during adolescence, EBV infection is associated with infectious mononucleosis, also called student‘s disease, kissing disease or in German Pfeiffersches Drüsenfieber.
Symptoms: Swallen lymph nodes -pharyngitis -Rash
Large, activated Lymphocytes are seen in the blood in high numbers during EBV infection (lymphocytosis)
The incubation time is 4-6 weeks. Symptoms like fever typically last for 2-4 weeks. The feeling of tiredness may last for months. The swollen spleen may cause splenic rupture: don‘t do sport during IM!
Describe the Moa of Foscanet
Zusatz VL4
competitive inhibitor of pyrophosphate
non hydrolyzable
bound by pyrophosphate binding site of the viral DNA polymerase
no cleavage, no energy for the reaction
back up therapy for acyclovir resistance, but high side effects
VL5 Papillomaviruses
What HPV subtypes can cause warts?
VL5
non risk HPV subtypes
e.g. HPV-1, 2
Plantar warts (Dornwarzen) are caused by non-risk HPV types (e.g. HPV-1, HPV-2, HPV4, PHV-60, HPV-63, and others). Vaccination against high-risk HPV does not protect against warts.
What HPV subtypes cause condyloma (Feigwarzen)
low risk subtypes (that cant cause cancer)
HPV-6, 11
What HPV subtypes cause cervic carcinoma?
HPV16 and HPV18
What percentage of PHV infection of the cervix results in viral persistence and cancer
when infected with high risk subtype: 10% persistence (integration into cellular genome) and 0.8% cancer formation
Approximately 0.8% of high-risk HPV infections lead to cancer
viral persistence 10%
How can HPV can cause benign tumors?
nfection of upper epidermal cells
E6 and E7 stimulate cell cycle entry from G0 to G1 in the upper epithelial cells
________________________
HPV triggers cell proliferation also in upper layers -The E6 and E7 proteins stimulate cell cycle entry from G0 to G1 in upper epithelial cells. A benign tumor (e.g. wart) forms.
The E6 and E7 proteins stiulate cell cycle entry from G0 to G1 also in basal cells. A malignent tumor (e.g. cervix carcinoma) forms.
HPV E6 and E7 inhibit the tumor-suppressors p53 and Rb. This causes cell proliferation and benign tumors.
How can HPV can cause cancer
E6 and E7 proteins stimulate cell cycle entry from G0 to G1 also in basal cells → malignant tumor
E6 and E7 inhibit immune response
infection of basal cells consisting of stem cells
aging skin cells enter the upper layers of the epidermis → virions are released
HPV infects the basal layer that consists of stem cells. Normally, the basal layer is the only zone in the epidermis with cell proliferation. As the aging skin cells enter the upper layers of the epidermis, viral gene expression occurs within them, and virions are released from the upper layers of the epidermis.
...know why HPV integration is key to cancer
the E6/E7 mRNA of episomal viral genome is unstable due to a AUUUA sequence → little/no Rb and p53 inhibition
if the genome becomes integrated into basal cells the structure of the mRNA changes as the unstable AUUUA sequence is not present
...know the targets of E6 and E7 (p53, Rb)
E6 → p53
E6 supports proteosomal degradation of p53 via E6 AP (E6 associated protein → ubiquitin ligasse)
this inhibits apoptosis
E7 → pRb
Rb usually binds to E2F and inhibits it
if E2F is active it stimulates the expression of S phase specific genes
this inhibits cell cycle progression
How can high risk HPV infection can be prevented
by vaccination
HVP16 and 18 are responsible for 70% of the cervical carcinomas
vaccine contains protection from these 2 risk groups and additional HPV that cause 90 % of all condylomas and 90% of all cervical carcinomas→ risk significantly reduced´
Why does the vaccine does not protect against CC (cervical carcinoma) if infection is already persistent and why
The vaccination protects only if there is not yet a natural, persistent infection
vaccine contains capsid protein L1
Tumorigenesis occurs via the E6 and E7 proteins of the HPVs, against which the vaccination does not build up an immune response, because it contains only capsid protein L1.
VL6 ss(+) RNA viruses
What is a (+)ss-RNA virus is
VL6
with mRNA sense genome
virus which contains a RNA genome that can be directly recogniced by ribosomes and translated into proteins after infection
Name examples for +ssRNA viruses
Rhinoviruses (Picornaviridae)
Yellow fever virus (Flaviviridae)
Noroviruses (Calciviridae)
Rubellla virus (Toga viridae)
Coronaviruses (Coronaviridae
What are essential components of rthe ssRNA genome for it to be translated?
3’ PolyA tail
5’ Cap
Can RNA virus genomes be directly translated by the ribosomes?
yes
Describe an experiment to prove that RNA itself is infectious
..know that isolated +ssRNA genomes are infec%ous amer transfection
Isolate RNA from the virus
add isolated RNA on a cell culture
new virus particles are created
What is the difference between monocistronic and polycistronic genomes. Name example viruses
monocistronic genome RNA
e.g. in Flaviviridae, Picronaviridae
synthesis on polyprotein from a single ORF
polyprotein is cleaved to multiple proteins by viral and cellular proteases
Polycistronic genome
e.g. in Astro-, Calci- or Togaviridae
multiple ORFs
first ORF translated from the genomic RNA and encodes the viral RdRp
subsequent ORFs translated from subgenomic mRNAs
subgenomic mRNAS are transcribed from the antigenome
Where does the replication of RNA viruses take place?
in the cytoplasm
on intracellular membranes - multivesicular bodies
proliferation and restructuring of membranes by viral proteins
membrane associated nonstructural proteins
in mutivesicular bodies, RNA is prtected from recognition by PRPs
viral and cellular proteins constrained at the required location
What enzyme activity is needed for genome replication, is ith part of the virus particle?
...need RdRp for replica%on, but it’s not part of the virion par%cle (in contrast to -ssRNA viruses)
RdRp needed
RdRp creates antigenomic RNA from genomic one and genomic from the antigenomic RNA
in comparison to (-)ssRNA it is not part of the virus particle
What is homologous recombination and what is its mechanism?
...homologous recombina%on and its mechanism and its .. ?
when a cell is infected by two viruses, template switching occurs ( the RdRp changes in between RNA templates and resumes synthesis if sequences are similar
creates new viruses with chimeric genome
only occurs in + RNA viruses but is the most important mechanism for the evolution of these viruses
What is an IRES? What are the differences between type 1 to 3? (no molecular details)
internal ribosome entry site
serves cap independent translation initiation → each ORF preceded by an IRES is efficiently translated
extensive secondary structures of RNA
type 1 and 2 IRES: eIF4G mediates binding of eIF3- 40S complex to IRES
type 3 IRES: 40S subunit binding requires only eIF3
Why is polyprotein processing needed? Which protein mediates it?
...know that monocistronic genomes need protease-mediated polyprotein processing
creates single proteins in monocisronic RNAs
initial cleavage cotranslational → never full polyprotein observed in cells
cleavage performed by viral proteases (initially 2A pro, then 3Cpro/3CDpro)
Draw the particle structure of corona viruses
spike glycoprotein timer (S)
Nucleoprotein (N) and RNA genome
Membrane protein (M)
Hämaggluthinin Esterase (HE)
envelope small membrane protein pentamer (E)
⇒ enveloped virus particle with large (30 kb) ss (+) RNA genome; 2/3 of genome codes for proteins of the polymerase complex, only RNA dependent RNA polymerase with proofreading
What is the function of SARS-CoV2 receptor ACE2
angiotensin-converting enzyme 2
entry receptor that binds to the spike protein
expression in the respiratory epithel of upper airways by Pneumocytes and Alveolarmacrophages, blood vessels and the kidney
cofactor TMPRSS2
What is Vpg (Viral protein genome-linked)?
Zusatz VL6
Example 1
as an exception, in picornaviruses RNA replication is dependent on a primer
Vpg serves as a primer
3CD assembles on CRE element and Vpg binds; Vpg is then uridylylated by 3Dpol which turns it into a primer→ binds to polyA tail
Example 2
in noroviruses or sapoviruses
Vpg serves as a cap replacement
⇒ hier könnte man wenn man Zeit hat noch den genauen Mechanismus verstehen
What is the genome composition in Noroviruses?
3 ORFs
ORF1: polyprotein encoding protease, polymerase and Vpg
ORF2: major capsid protein VP1
ORF3. smaller structural protein
ORF2 and 3 expressed via subgenomic mRNA
Name members of the coronaviridae family.
alpha, beta, gamma and delta coronaviridae
SARS coronavirus is a beta coronavirus
How does transcription and replication in coronaviruses take place?
discontinuous transcription and genome replication
replication in double membranous vesicles derived from the ER and restructurd and formed by viral proteins
Describe the replication cycle of corona viruses.
fehlt
VL7 ss(-) RNA Viruses
What is a (-)ss-RNA virus?
VL7
virus with non infectious RNA associated with functional polymerase complex
all enveloped
max 19 kb genome
transcription via sequential stop-start mechanism
Name 3 examples for (-) ssRNA viruses
...know measles virus, influenza virus, RSV as examples for -ssRNA viruses
measles
Influenza
RSV
Does the virion contain a RdRp?
What are its minimal transcription activities? Zusatz
...that virions require RNA-dependent RNA Polymerase
minimum transcription of Large protein L, Phosphoprotein P and nukleoprotein N or NP
Are isolated -ssRNA genomes infectious?
...know that isolated -ssRNA genomes are non-infec%ous amer transfection
no
What is a segmented and non-segmented genome?
non segmented: e.g. Rhabdoviridae
segmented: e.g. Orthomyxoviridae (influenza)
in influenza 8 (-) ssRNA segments
each segment encodes for a single gene
M and NS segments code for two proteins by splicing of mRNA
segmented negative and ambisense: e.g. arenaviridae
where does replication of ssRNA viruses take place?
...replication of almost all -ssRNA viruses in cytoplasm
cytoplasm except Bornaviridae
What is the influenza entry receptor?
...influenza receptor sialic acid
(N-acetyl-neuraminic/) sialic acid
⇒ hemagglutinin is selective concerning the galactose links of the sialic acid
What are the influenza surface antigens?
...influenza surface antigens H and N
Hemagglutinin (H)
and Neuraminidase (N)
also important M2 transmembrane protein
Name properties of Hemagglutinin and Neuraminidase
VL 7
Hemagglutinin
trimer
binds sialic acid
name comes from ability to agglutinate erys
fusion activity is pH dependent and requires proteolytic cleavage
main target of neutralizing antibodies
Neuraminidase
tetramer
cleavage of terminal N-acetyl neuraminic acid → receptor destroying enzyme
NA activity needed when virus leaves the cell
also target for antibodies
Is the RNA tightly associated with N?
Zusatz VL7
yes, in comparison to +RNA viruses whee only a loose contact is needed to allow homologous recombination, here the RNA must be tightly associated with N
Explain the sequential start-stop mechanism of mononegavirales
requires L, P, N and M2-1
M2-1 is unique in Pneumoviridae
M2-2 inhibits replication (also unique)
RNA consists of segments in between 3’ leader and 5’ trailer
the segments are seperated by gene start and gene end sequences
RNA polyermase slipps over U7 sequence, termination of mRNA after 200 A’s are added
initiation and capping of next mRNA
polymerase always binds to 3’ end
3’ end genes are more transcribed → transcriptional gradient
Give an overview of the genome composition in negavirales. (might be incomplete)
all except Pneumovirus have a 3’ N and 5’ L protein
3 intramembrane proteins
F protein
G glycoprotein
SH protein (ion channel)
2 Matrix proteins
major: M
M2 product of first ORF
How does switch from transcription to replication take place in Pneumoviridae?
via accumulation of N and M2-2
packaging of replicative viral (+) RNA in N
3’ and 5’ are complementary
if the polymerase transcribes the whole gene, the new replication cycle starts
the trailer complement is recognized as a promotor
How does influenza entry take place?
acidification of the endosome
acidification of the virus particle (via ion channel M2)
conformational change in hemagglutinin that was previously activated by proteolytic cleavage leads to insertion of the fusion peptide into the endosomal membrane
fusion of viral and endosomal membrane
release of RNPs into the cytoplasm
transport of RNPs into nucleus
transcription and genome replication in nucleus
What is cap-snatching?
name explains itself
PB2 binds host 5’cap
PA cleaves after A
snatched cap acts as a primer for +strand synthesis from the - strand genome
VL8 ARI and GI
Name important acute respiratory infection viruses (ARI)
VL8
...important ARI viruses: influenza, Corona, RSV, Adeno, HMPV, PIV, rhino, coxsackie
corona
Adeno
HMPV
PIV
Rhino
Coxsackie
Name symptoms of ARIs
...symptoms: respiratory tract symptoms include rhinitis, pharyngitis, bronchitis, pneumonia
rhinitis (Nasenschleimhautentzündung)
bronchitis (Entzündung der Schleimhaut in den Bronchien)
pneumonia (Lungenentzündung)
Name seasonality of viruses. When do they occur?
winter: influenza, Corona, RSV
summer: coxsackie
virus all year: all others (Adeno, HMPV, PIV, rhino)
spring hMPV
spring/fall Rhino
all year: all others (adeno, PIV)
What virus causes severe respiratory infections and hospitalization in children under 2 years?
...RSV cause of severe respiratory infections and hospitalization in <2 year old children
What is influenza antigen drift and shift
shift:
introduction of a new HA and/or NA subtype in the immunologic naive population
only influenza A virus
Reassortion or direct transmission from e.g. avian viruses to humans
⇒ causes pandemics
Drift:
Small/single point mutation in HA and NA that gradually modify epitopes and thus antigenicity
e.g. in HA timer → 5 epitopes/ monomer→ each mutation can destroy epitope
⇒ causes seasonal epidemics
...mechanism of reassortion
basis: segmented genome
prerequisite: infection of a cell by two different influenza A viruses
exchange of genome segments and thus genes
reassembly of virus with new properties
reassortants of HA or NA segments: no crossreactivity with existing antibodies
What causes severe respiratory infections and hospitalization in old people?
...influenza cause of severe respiratory infections and hospitalization in old people
What is the natural reservoir of influenza A? Which animal serves as a mixing vessel to spread the virus to humans?
...influenza A: natural reservoir is poultry, pig as “mixing vessel”, spread into human population
natural reservoir: poultry (Geflügel)
mixing vessel: pig
What is the host of influenza B?
...influenza B: humans are the only hosts
...influenza: what does H1N1 or H5N1 mean?
Influenza A virueses are destinguished depending on the subtypes of the surface proteins hemaggluthinin (H) and neuraminidase (N)
18 HA subtypes (H1,2,3 found in humans)
11 Na subtypes (N1,N2 found in human)
H1N1 → swine flu pandemic, russian flu and spanish flu
H5N1 → bird flu/ avian influenza
⇒ all subtypes found in aquatic birds (main reservoir)
Name GI viruses
Valentin 18 Zusatzfolien zu VL8
—> compare if necessary
rotaviruses
Calciviruses
Astroviruses
Coronaviruses
Name common properties of Enteropathogenic infections.
Zusatz VL8
non enveloped viruses
highly resistant against environment
particles infectious for extendet period outside the host
viruses highly contagious
transmission fetal oral
replication restricted to proximal small intestine
massive virus shedding in stool
Why is research of respiratory and gastrointestinal infections important?
lower respiratory tract and diarrheal infections under top 10 causes of death world wide
and top 2 causes of death in low income countries
LRTI range 5th in death causes of eldery and 2nd in that of childrean
Name a LRTI
laryngitis subglottica = Croup → Kehlkopfentzündung → ptentially fatal
What is the surface and ventilation rate of the LRT?
surface 140 m^2
ventilation rate of 6l/min
⇒ most common portal of entry
Give an example how targeted particle release determines tropsim?
Vl 8 Zusatz
apical release:
influenza
⇒ local infection
basal release
VSV
⇒ systemic infection
State reasons for seasonality of viruses
Name properties of syncytial virus
forms sycytiain certain celllines
endemic infection about 90% at the age of 2
highly contagious
seasonallity: Oct. till April
risk patients
childrean with cardio pulmonary diseases
elderly → significant morbidity and mortality
hematologic patients
What is the transmission route of RSV and what are the symptoms?
Transmission:
droplets, smears and aerosoles
primary site of infection: nasopharyngeal epithelium
spread in lower and small airways
incubation 2-8 days
Symptomes
rhinitis (nasenschleimhautentzündung), Cough (Husten), bronchitis, hyperventilation, wheezing( keuchen), cyanosis (blaufärbung haut und Schleimhaut)
severe infection: Bronchiolitis → infection lower respiratory tract
What is the pathogenisis of influenza?
transmission via aerosols
epidemics in winter
tracheobronchitis = infection without complications
incubation time 1-5 days
abrupt onset with high fever that eases after 2-3 days
unwohlsein und erschöpfung up to 2 weeks
Name influenza complications
primary pneumonia
superinfection with bacteria and pneumonia
further complications: Otitis Media, Myositis, Myocarditis, Ecephalopathy
Lethality 0.2% to 2.5% (1918)
people with increased risk: eldery, immunocompromized, pregnant
What is the difference between high pathogenic influenza virses and low pathogenic ones?
low pathogenic:
HA cleaved/activated extracellularly by trypsin like airways proteasee
only distinct areas of the GIT and the RIT can be infected
highly pathogneic
HA cleaved intracellulrly
systemic infection
Name properties of Rotaviruses
family Reoviridae
high environmental stability
wheel like structure
fecal oral transmission
seasonaltiy in winter
dsRNA, (+) strand with 5’ cap
virion consists of 3 layers with distinct functions (infection, transcription, replicatio)
11 segments,monocistronic → reassortion possible
symptomes: fever, vomiting, Diarrhea, dehydration and loss of electrolytes
symptoms for average of 5 days
⇒ globally most significant cause of severe gastrointestinal infections in childrean under 5
Explain the replication cycle of Rotaviruses
binding to receptor and uptake by receptor mediated endocytosis
pH dependent fusion associated by shedding (ablegen) of VP5 and VP8; release of DLP (double layered particle) into cytoplasm
transcription in DLPs; mRNAs extruded from pores of the DLPs; translation of mRNAs
Translation of NSPs leads to formation of viroplasm
amount of new VP1 and VP3 induces the switch to repcliation
VP1 and 3 bind to 3’ end of mRNAs and trggers recruitment of VP2 → core formation: mRNA is copied to (-) RNA → ds foramtion
VP6 assembles onto nascent core → DLPs
NSP4 asequesters VP4 and DLPs via VP6 to the cytosolic surface of the ER; budding into ER forming a transietly enveloped particle
formation of TLP by removal of ER membrane and NSP4; release of TLP by exocytosis or lysis
Describe the pathogenisis of Rotaviruses.
fecal-oral transmission
infection of mature enerocytes in small intestine
cytopathic for infected cells: lysis for virus escape and shut down of protein synthesis
parakrine cytopathic effect on non infected cells: enterotoxin NSP4 mobilizes Ca2+ and permeabilizes tight junctions
Name Rotavirus vaccines
Rotarix
live attenuated
monovalent
oral administration
derived from human strain attenuated by sequential passages
RotaTeq
pentavalent
based on reassortment of bovine strains
VL9 Infektion Epidemiology
Definitions of endemic, epidemic, pandemic
VL9
endemic: normal/usual occurence of a certain infectious disease in a certain population
e.g. influenza/ SARSCoV-2 in 2022
epidemic: unusally stong and temporally limited occurrence(accumulation of a certain infection disease in a certain area
e.g. ebola in west africa in 2014
Pandemic: an endemic of a new pathogen that spans countiers/continents
e.g. SARS-CoV2 in 2019-2020
Definitons of prevalence, incidence, morbidity, mortality, CFR
prevalence: proportion of infected people in a population
e.g. prevalence of HIV infections in Germany is 0.1%
incedence: number of new infections in a population in a certain time span
e.g. incidence of HIV infections in Germany is 3500 cases per year
morbidity: proportion of people who get sick in a population caused by the the infectious agent
e.g. 24.000 disease cases per 100.000 people
mortality: proportion of people who die in a population caused by the infectious agent
e.g. 120 deaths per 100000 people
CFR (case fatality rate) or lethality: proportion of people who die among people with diagnosed infection
e.g. 9000 deaths per 300000 diagnosed infections =3%
How to calculate prevalence, mortality, CFR from numbers
Difference of Weekly and cumulative incidence
weekly incidence shows number of cases per week
cumulative incedence shows total cases over time → adds up number of new cases
at first look more impressive, good to see which proportion of individuums already hat contact with the pathogen
incedence of 0 looks like a horizontal line
What is R0 and what are R0 values of Measles, polio and SARS?
R0: baseline reproduction rate
average number of subsequent infections caused by one case of infection in a population that is immunologically naive to the pathogen
usually calculated from increase in incidence numbers in the early weeks of a pandemic or epidemic
e.g. Masern R0=12-18, Polio 5-7, SARS/HIV 2-5
What is herd immunity?
VL 9
herd immunity is archived when a sufficiently high proportion of the population has become immune to a pathogen through infection or vaccination so that the incidence of the new infections declines
if reached pathogen can no longer spread
but if herd immunity is reached and the pathogen has enough genetic flexibility, immune escape variants wil emerge
What is achieving herd immunity dependent on?
archievin herd immunity is dependent on:
contagiousness of the pathogen (R0)
duration of immunity
pathogen variability and its ability to generate immune escape muations
What is R0 and herd immunity? How can you calculate herd immunity from R0?
With an R0 of 5, what proportion of the population has to be vaccinated/infected to achieve herd immunity?
0,8 → 80%
What is the effective reproduction rate?
Zusatz VL9
average number of subsequent infections caused by one case of infection in a population that has already built up immunity to some degress
Re decreases during epidemic or pandemic because immunity increases
Is there a Seasonal varia%tin of R0 and herd immunity for respiratory infections? What effect does this have on herd immunity?
most respiratory viruses have a higher R0 in winter and a lower R0 in summer
therefore also herd immunity oscillates → R0 reduced in summer compard to winter by 40%
Why is R0 often overestimated at the beginning of a pandemic?
Know that there is a Seasonal varia%on of R0 and herd immunity for respiratory infections
What does the SIR-model do (just very briefly what it does, no math)
Why is it oversimplified?
Susceptible, Infected, resistant model
susceptibe nuber decreases, infectios peak and resistant number increases
oversimplistic but useful model forthe first weeks of an endemic
doesnt respect decreasing immunity
doesnt inculde seasonal variation of R0
does not include changes in behavior of population
does not include effects of microenvironments like kindergardens or eldery homes
no long term predictions possible
What are Excess deaths and how to calculate excess deaths
how many more people died in a certain week of a year compared to the average number in the same week of the last few years
mostly reference period of last 4 years used
calculation: (number of deaths within a week)- average number of deaths in this week of the past 4 years)
What is RRR, ARR, NNT and calcula%on of these values from a 4-field analysis
event = get sick
RRR= relative response rate: good index for effectiveness when you are already infected
RRR= (0.03-0.05)/0.05= -0.4 → treatment reduces the risk by 40%
ARR=absolute response rate: goog for profilaxe → e.g. malarone → chance to get malaria in germany super low in the first place
ARR=(0.03-0.05)= -0.02 treatment reduces the absolute risk by 2% points
NTT= number needed to treat
NTT= 1/ARR= 1/0.02=50 → 1 of 50 patients profits from treatment
When is RRR relevant when ARR?
RRR is a good parameter for treatment
ARR is a good parameter for prophylaxis
VL 10 Antiviral Immunity
What is intrinsic, innate and adaptive immunity?
VL10
intrinsic immunity: immune response within a single cell
Innate immunity: complex immune response directed at specific surfaces
adaptiv immune response: complex immune response directed at variable structures
Assign the different immune responses to innate, intrinsic and adaptive
intrinsic:
TLRs, interferon, tetherin, apobec3G, epigenetic silencing, induction apoptosis
Innate:
cellular components (granulocytes, mactrophages, NK cells)
humoral components (complement, interleukins)
Adaptive
B and T cells
How can receptors form intrinsic immunity recognize foreign structures?
Know how receptors from intrinsic immunity can recognize foreign structures (unique ligands or unique loci)
unique ligands or unique loci
inhibition of transcription
inhibition of 5’ end dependent translation
Inhibition of ER-to Golgi traffic
Syncytium formation
Inhibition of transport of host RNA
Depolymerization of the cytoskeleton
Name the 4 TLRs important for antiviral defense and their ligands
Tlr3. dsRNA
Tlr7/8: ssRNA
Tlr9 CpG DNA, unmethylated CpG oligonucleotides
⇒ recognize only foreign ligands by only recognizing unique loci or being active at unique locations→ main effector response is interferon and cytokine production
How can receptors make sure to only detect foreign structures?
some receptors recognize unique ligands
e.g. TLR3 and Mda5→ dsRNA
or Rig1→ RNA with 5’ phosphate (not present in capped mRNA but in HCV RNA)
Recognize ligands at unique loci
e.g. TLR3,7,8,9 localized in endosomes where usually no cellular DNA or RNA is present
Name 5 examples for intrinsic defense mechanisms and explain them
antiviral state:
triggered by interferon I
increased cellular concentration of dsRNA-activated protein kinase Pkr in uninfected bystander cells
if these cells become infected with dsRNA viruses, Pkr becomes active and shuts down translation by phosphorylating and thus inhibiting eIF2a
tetherin
interferon induced
inhibits release of enveloped viruses by sticking into the membrane bilayers
counteracted by HIV-1 vpu protein
APOBEC3G (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G)
deaminates C to U in ssDNA → this hypermutation introduces multiple stop codons
HIV-1 vif counteracts APOBEC3G by degradation of A3G
epigenetic silencing
silencing of new DNA in the nucleus by methylation of DNA or deacetylation of histones
apoptosis
infection of cells with virus increases apoptosis rate for many viruses
many viruses counteract this by encoding antiapoptotic proteins
cell surface: production of apoptosis inducing cytokines; alteration of membrane integrity
cytoplasm: translation inhibitiors, modification of cytoskeleton, disruption of signal transduction
Nucleus: DNA damage, altered gene expression, diruption of the cell cycle
What is the ultimate effect of TLR signaling?
Know that TLR-signaling ul%mately results in Interferon I production
interferon 1 production
Which cytokine can trigger an antiviral state?
Know that Interferon I can trigger an antiviral state
interferon I (alpha and beta); no direct antiviral effect but activation of antiviral pathways; cause flu like symptomes
Know what „antiviral state“ means
IFNa leads to increased cellular concentrations of dsRNA-activated protein kinase Pkr in uninfected bystander cells
if these cells are infected with dsRNA, Pkr becomes active and shuts down translation by phosphorylating eIF2
Know about MHCI and MHCII and what they do /molecules they interact with / what is their function
MHC1:
expressed on all nucleated cells (all cels except erys)
presents antigens to CD8+ T cells (CTLs)
predominantly loaded with self antigens that have been expressed in the same cell; alternatively MHC1 can also be loaded with self antigens that have been taken up → cross presentation
loading results in priming of naive CD8+ T cells by unifected APCs or killing of infected MHC-i presenting cell
signals the immune cell. kill me
MHC2:
expressed on APCs (DCs, Macrophages, microglia, B cells, T cells and epithelial cells)
presents antigen to CD4+ T cells(helper T cells)
predominantly loaded with self antigens that have been taken up, alternatively MHC1 can also be loaded with endogenous self antigens → signals helpme
NK cells kill other cells that dont express enough MHC1 (missing self)
MHC1 inhibits the NK cell from killing
killing by perforin and granzymes
many viruses cause downregulation of MHC1
Know what cross-presentation means
Clara:
MHCI ist normalerweise mit Antigenen aus der Zelle beladen MHC I mit Antigen außerhalbder Zelle
Cross penetration Ist die Fähigkeitvon Dendritischen Zellen DC MHC I Moleküle mit Antikörper zu beladen welche aufgenommen wurde Cexogeneaus antigens
ability of DCs, macrophages and B cells to load MHC1-molecules with antigens that have been taken up; usually MHC1 is only loaded with antigens that have been expressed in the same cell
this is an important mechanism for the activation of naive CTLs to effector CTLs → only a few viruses infect DCs but all have to be recognized by CTLs
How are B cells activated to produce antibodies?
Know how B cells are activated to produce antibodies (Summary B cell immune response)
viral antigen taken up by naive B cell via BCR
B cell migrates to lymph node and presents antigen via MHC2 effector TH cells
Th cell recognizes antigens presented by MHC2 on B cells and releases activating cytokines
B cell develops into long living plasma cell that initially produces IgM and later IgG
some of these activated B cells develop into long lived memory B cells
upon secondary infection memory B cells can quickly develop into new plasma cells and produce high affinity IgG( no need for stimulation by T cells)
How are T cells activated?
Know how T cells are activated (Summary T cell immune response)
viral antigen taken up by DCs
Dc presents parts of the antigen on MHC2 and (by cross presentation) on MHC1
Dc migrates to lymph node and presents antigen to naive T cell
a. an antigen specific naive CD4+ cell recognizes the antigen and develops into a effector TH cell
b. an antigen specific CD8+ T cell recognizes the antigen and develops into an effector CTL cell
effector TH and CTL migrate to site of inflammation
CTL recognizes infected target cells by antigen presented on MHC1 and kills the cell
TH cell recognizes antigens presented by MHC2 on APCs in the tissue and releases activating cytokines
most of the effector CTLs and TH cells die after 2-3 days by activation induced apoptosis
some cells survive and become long living memory T cells
upon secondary contact with the same virus these memory cells can quickly develop into new effector cells
Know how viral infections can cause autoimmunity, including myocarditis
B cells that recognize autoantigens may incorporate viral antigens during a viral infection and present these viral antigens on MHC2; viral specific TH cells can then stiummlate the autoreactive B cell to produce autoantibodies
often autoimmunity in the heart resulting in myocarditis and pericarditis triggered by cardiotrophic viruses like Cocksackie, influenza or SARS-CoV-2 (also triggered by covid vaccines)
___________________
Viren induzieren Autoimmunität B Zellenerkennt körpereigenen Antikörper
B Zellen welche Autoantigene erkennt kann virale Antigene während einer viralen Infektion und präsentieren diese viralen Antigene auf MHCII Zellen können die autoreaktive B Zellen aktivieren um antikörper produzieren
VL11 Antiviral vaccines
... the different vaccine types (live, vector, mRNA, dead, protein, conjugate, polysaccharide), their components and their basic mechanism
VL11
Klausur
life vaccines
attenuated, replication competent viruses
e.g. MMR, influenza
vector vaccines
replication incompetent transgene viral vectors(e.g. adenoviral vectors), that express one gene of the pathogen
e.g. SARS CoV2, Ebola
RNA vaccine
N1 methyl-pseuouridine-modified mRNA, packaged in lipid nanoparticles , adjuvants
e.g. SARS CoV2
dead vaccine
formaldehyde-inactivated virus, adjuvans Al(OH)3 or AlPO4
e.g. Polio, Influenza(for influenza proteins seperated from virus → split vaccine)
protein vaccine
protein antigen, adjuvans (Al(OH)3 or AlPO4 or saponins
e.g. SARS CoV2, HBV
conjugate vaccine
polysaccharide-antigen coupled to protein, adjuvans (Al(OH)3 or AlPO4)
e.g. Memingococcus, pneumococcus
polysaccharide vaccine
polysaccaride-antigen, adjuvans(Al(OH)3 or AlPO4)
e.g. pneumococcus
...what an adjuvant is and why it is needed in some vaccines
substances added to vaccine to increase the immunogenicity
activation of inflammasome → proinflammatory, acitvation of TLRs
e.g. aluminium hydroide or Aluminium Phosphate
can increase side effects → fever bone pain, pain at injection site etc.
lipid nanao particles, modified RNa from mRNA vaccines and particles of vector vaccines (e.g. adenoviral particles) have a proinflammatory adjuvant effect
...know the different effector functions to kill virus-infected cells
cytotoxic T cell killing
complement dependent cytotoxicity
antibody dependent cellular toxicity (ADCC)
antibody dependent cellular phagocytosis
...know how the covid mRNA vaccines work
N1-me-pseudouridine modification increases mRNA stability but decreases TLR signaling (low intrinsic adjuvant activity)
Lipid nanoparticles (LNPs) act as a carrier system for enhanced transfection end endosome escape and also as adjuvants
mRNA vaccine for covid contains mRNA coding the spike protein S1
⇒ in vivo transfection with mRNA encoding the antigen
...know what a prime/boost scheme means in vaccination and what it does
during intital immune response to the vaccine(prime) low affinity IgM, IgG and IgA rapidly formed by extrafollicular activation
folicular maturation results in formation of hih affinity IgG and IgA after approximately 2 weeks
antibody titers may decrease again over time
booster vaccination leads to activation of memory B cells which quickly secrete high affinity IgG and IgA in large quantities
What are the different classes of antibodies?
VL 11 Zusatz
IgM
pentameric
B cell receptor
serologic signal for fresh infection or reactivation
production after first contact, usually disappears again
important for complement activation
IgG
monomeric
late production
important for neutralizing in plasma
titer stay up for long time
enters plazenta
production if antigen is in peripheral lymph nodes
IgE
IgA
like IgG active in plasma and monomeric
sIgA
dimer
important for neutralization of mucosa
late production when antigen is presented in mucosal lymph nodes (e.g. peyers patches)
Why is mucosal antigen delivery needed for sIgA response?
sIgA produced after presentation of antigen in mucosal lymph nodes
sIgA important for mucosal immunity
What are the three main principles of vaccines (life, antigen, gene-based)
injection of life attenuated virus
e.g. measles
same tropism as WT infection
infection route can be the same or different
mucosal tissue might be only secondary infected and protection afterwards can be weaker than the WT infection
injection of proteins, protein mixtures and polysaccharide antigens
dead vaccines
protein vaccines
konjugationvaccines
polysaccharide vaccines
Toxoid vacines
Wt infection only rearmed to limited extend
no tropism
direct uptake of antigen by APCs, limited production of mucosal immunity
in vivo transfection of nucleic acid (gene therapy)
mRNA vaccines
limited rearming of the infection
active component is a nucleic acid that is translated to a protein by the uptaking cell
limited mucosal immunity induction
What is a neutralizing antibody?
bind to viruses or bacterial toxins and thereby prevent interaction with their respective cellular receptor
What is the function of vaccination?
create an immunological memory that prevents sympotoms upon the first infection
What is the difference between Natural and adaptive immunity?
natural
fast after first contact
no immunological memory
specific for bacterial polysaccharides, dsRNA, bact DNA, missing self
by NK-cells, makrophages. interferons, complement, granulocytes, PRRs and TLRs.
triggered by adjuvans
slow after first contact
fast after second contact
immunolgical memory
highly specific for certain AS sequence
by B Cells, T cells and antibodies
triggered by vaccines
⇒ in between both are NKT cells, DCs and gamma delta T cells
Last changed7 months ago