Virologie

Susceptibility: The cell expresses a receptor for viral entry. The virus can enter the cell

Permissivity: The cell can multiply the virus and release new viruses

envelope: viral membrane

Envelope protein: viral protein embedded into viral membrane

capsid: viral protein core

nucleocapsid: capsid that directly interacts with the viral genome

pfu: plaque forming units

moi: multiplicity of infection

CPE: cytopathic effect

+ssRNA, -ssRNA, dsRNA, +ssDNA, -ssDNA, dsDNA: positive- or negative-oriented single stranded or double stranded RNA/DNA

ID50: infectious dose that infectes 50%

ccID50: ID50 in cell culture (cell culture infectious dose 50%)

LD50: lethal dose 50%; dose at which 50% of animals die

budding: release of viral particles through a cellular membrane and the acquisition of a viral envelope through this process

Virus-like particle: empty capsids that spontanously form from capsid monomers; used for vaccines

in vitro: - experiments with enzyme mix (e.g. in an Eppi), experiments in cell culture

in vivo: experiments in living organisms. E.g. animal experiments, treatment of patients

tropism: which cell type can become infected?

-> the presence of binding receptors on a cell determines the tropism of a virus: the virus can only enter cells that express this molecule. Cells that don‘t express this molecule cannot become infected

host range (Wirtsspektrum): which species can become infected?

RBD: receptor binding domain

FP: fusion peptide

Viral attachment: lose binding to attachment factors

Viral binding: binding to entry receptor

Affinity: binding strength between ligand and receptor

Avidity: sum of all affinities if multiple binding sites exist

adeno viruses

hepadna viruses

herpes viruses

orthomyxo viruses

picorna viruses

retro viruses

nido viruses

The replication cycle e.g.: Poliovirus

1. Attachment and Entry

2. Release of Genome (Uncoating)

3. Translation of viral proteins

4. Genome replication

5. Assembly

6. Release

7. (sometimes: maturation)

= fusion of cells (fusion of the viral envelope membrane with the cell membrane), mediated by viral envelope proteins, often seen as CPE in cell cutures infected with enveloped viruses

physical titer: measurement of viral particles (protein or genome; by ELISA or PCR)

biological titer: determined from infection experiments

-> physical titer is usually higher than biological titer

-> biological titer measures only intact/infectious particles and is also dependent on the type of cell culture being used

PCR with 2 primers and a probe

- Probe carries fluorochrome and quencher

- Taq Pol has exonuclease activity and digests probe if it binds to PCR product

- Fluorochrome gets released from quencher and fluorescence is measured in a light cycler

receptor: viral entry

coreceptor: assists in viral entry, a second binding receptor that is needed to allow viral entry

attachment factor: lose attachment of virus to cell

•…endemic: normal occurrence of a certain infectious disease in a certain population

• …epidemic: unusually strong and temporally limited occurrence (accumulation) of a certain infectious disease in a certain area

• …pandemic: an epidemic of a new pathogen that spans continents/the whole world

• … prevalence: total number of infections in a population

• …incidence: number of new infections in a given time period

• …morbidity: number of disease cases in an entire population caused by a virus

• …mortality: number of deaths in an entire population caused by a virus

• …CFR: case fatality rate, number of deaths among infected individuals

• …prevalence: 504 infections/100.000 inhabitants

• …mortality: 27 deaths/100.000 inhabitants

• …CFR: 27 deaths/504 infections

• RRR: relative response rate, good parameter for treatment

• ARR: absolute response rate, good parameter for prophylaxis

• NNT: number needed to treat in order to prevent 1 disease case

• NNV: number needed to vaccinate in order to prevent 1 infection

4-field analysis: control treatment without disease, control treatment with disease, active treatment without disease, active treatment with disease (e.g. 90% versus 10%; 98% versus 2%)

Calculations from 4-field analysis:

• …RRR: (10%-2%)/10% = (0,1-0,02)/0,1 = 0,8 => treatment reduces risk by 80%

• …ARR: 10%-2% = 0,08 => treatment reduces risk by 8%-points

• …NNT: 1/ARR = 1/0.08 = 12.5 => 1 of 50 patients profits from treatment

• …NNV: 1/ARR = 1/0.08 = 12.5

Weekly incidence: the number of new infections per week is given

- If the number of infections increase over time, weekly incidence increases

- If the number of infections over time declines, weekly incidence decreases

• Cumulative incidence: the number of new infections in each week is added to the number of infections in the past.

- If the number of infections increase over time, cumulative incidence increases

- If the number of infections over time declines, cumulative incidence still increases

- If the number of infections over time is 0, cumulative incidence stays on a plateau

R0: basic reproduction number: average number of infections that are caused by 1 infected individual

herd immunity: is reached if a high-enough amount of people in a population is immune so that the virus can no longer spread in the population with a positive R

calculate herd immunity from R0? R0-1/R0

=> there is a seasonal variation of R0 and herd immunity for respiratory infections

SIR-model: Mathematical model to predict numbers of infections in a population: 3 key parameters. S = number of susceptible, I = numbers of infected, R: numbers of resistant/immune people

• SEIR-model: better mathematical model to predict numbers of infections in a population: 4 key parameters. S = number of susceptible, E= exposed but not yet infectious, I = numbers of infected, R: numbers of resistant/Immune people

• Excess deaths: more deaths than usually expected

how to calculate excess deaths: example: (deaths per week of this year)-(averagenumber of deaths of the same week of the last 4 years)

• Serial interval: describes the average time between the infection of a patient and the transmission to the next patient

• “flatten the curve”: Attempt to slow down infection spread in a population, e.g by wearing masks etc. Total number of deaths/illness until herd immunity is reached is not changed, but peak numbers are lower. Concept to protect hospitals from overcrowding

HAV, HBV, HCV, HDV, HEV

chronic disease —> HBV, (HDV),HCV

transmission:

—> Sex: HBV, HDV, HCV

—> smear infection/oral: HAV,HEV

—> drug abuse: HBV, HCV

prevented by vaccination: HAV, HBV, (HDV)

—> virus contains partly dsDNA,

—> DNA is completed in the cell,

—> cccDNA persists as episome in nucleus,

—> genomic RNA transcription from cccDNA,

—> reverse transcription of DNA genome from genomic RNA,

—> DNA incorporation into new particles,

—> ds-DNA synthesis stops in virus particle if all nucleotides are being used up

HIV: RT immediately after infection: gemonic RNA -> cDNA (early)

HBV: RT late in replication prior to exit: genomic RNA -> genomic DNA (late)

• How can HBV infection be treated? Nucleoside analogs (RT-inhibitors)

-> +ssRNA genome, gets directly translated at ribosome after infection

-> dsRNA, new +ssRNA genomes transcribed from the dsRNA, incorporation into new particles, nucleus not involved

treatment: Nucleoside analogs, protease inhibitors, NS5B-inhibitors

=> HCV has no mechanism of persistence. If replication is blocked, the virus is gone. HBV has a mechanism of persistence: ccc-DNA persists lifelong in nuclei of hepatocytes

similarities: +ssRNA

differences: no cap but IRES, no poly A but UTR

IRES: Initiation of translation; mimics the function of the 5’ cap of mRNAs

in molecular biology: IRES allows expression of 2 different proteins (not a fusionprotein) under control of one promotor: promotor-geneX-IRES-geneY

Retrovirus (RT): virus with RNA genome, integrates into host genome

Endogenous retrovirus: retrovirus that has entered the germline. Some endogenous retroviruses have lost their ability to replicate

PBS: primer binding site

LTR: long terminal repeat

Provirus: retroviral genome integrated into host-genome

retroviral replication cycle: virus particle with RNA, entry into cell, release of RNA, RT of RNA into cDNA, integration of cDNA into host genome (provirus), transcription of provirus: translation of viral proteins and transcription of genomic RNA, assembly andpackaging, budding at cell membrane

3 enzyms of reverse transcriptase

• RNA-dependent DNA polymerase

• DNA-dependent DNA polymerase

• RNaseH

in vivo: RNA template, RT, tRNA as primer, nucleotides

in vitro: RNA template, RT, DNA primers, nucleotides

-> RNA genome, tRNA primer binds at primer binding site PBS near the 5’ end of the viral genome -> synthesis of first cDNA strand by RT

-> degradation of RNA template (RNaseH activity) in DNA/RNA heteroduplex by RT,

-> synthesis of second cDNA strand using parts of the original RNA template as primer and removal of RNA primer by RT

HIV protease: Cleavage of Gag proteins to allow maturation of viral particles

HIV integrase:Integration of HIV cDNA into host genome

productive infection: provirus is transcriptionally active so that new viral particles are being produced

latent infection: provirus is transcriptionally inactive and no viral particles are being produced

reactivation: switch from latent to productive infection

HIV pathogenesis: HIV infects CD4+ T cells, depletion of CD4-T cells over time, after 10 years (on average) AIDS

therapy: combination therapy of different antiviral substances (RT inhibitors, integrase inhibitors, protease inhibitors)

SARS-CoV-2: Covid19

Rhinoviruses: common cold

HAV: hepatitis

HCV: hepatitis

-> RNA genome is infectious because it can directly be translated at the ribosome

-> replication involves dsRNA because +RNA replication requires a -RNA template strand

-> homologous recombination: template switching after coinfection

(+) RNA: can directly be translated at the ribosome into proteins

• mRNA: cap, UTR, ORF, UTR, poly A

• viral +ssRNA: often no cap, but: VPg/FAD and IRES, sometimes no poly A

• function of VPg or FAD: Substitute for mRNA-cap, examples for viruses that require VPg (picornaviridae, caliciviridae) or FAD (flaviviridae)

• function of the furin cleavage site in SARS-CoV-2: Cellular furin proteases cleave S2 protein

• +ssRNA-viruses need RNA-dependent RNA polymerase for replication, but it’s not part of the virion particle (in contrast to -ssRNA viruses) because it can be directly translated from +ssRNA genome

• SARS-CoV2 receptor: ACE2

-> Mononegavirales = -ssRNA viruses with non-segmented genome

-> RSV (lower respiratory tract disease)

-> parainfluenzavirus (respiratory disease)

-> measles virus (measles)

-> mumps virus (mumps)

-> -ssRNA genome must be first complemented to dsRNA to allow synthesis of mRNA

• virions require: RNA-dependent RNA Polymerase

• isolated -ssRNA genomes are non-infectious after transfection

• RSV (respiratory syncytial virus) is the most common cause of LRTI in children <2years

• important ARI (acute respiratory infections) viruses: influenza, Corona, RSV, Adeno, HMPV, PIV, rhino, coxsackie

• symptoms: respiratory tract symptoms include rhinitis, pharyngitis, bronchitis, pneumonia

• seasonality of viruses

-> winter: influenza, Corona, RSV

-> summer: coxsackie virus

-> all year: all others (Adeno, HMPV, PIV, rhino)

A: aquatic birds

B: humans are the only host

influenza antigen drift and shift

-> antigen drift: point mutations in the genome

-> antigen shift: reassorting

• What is needed for antigen shift = mixing of genetic material between viruses with segmented genomes during replication when they infected the same host cell, producing new combinations in the offspring viruses

-> influenza genome is segmented;

-> infection of one cell with 2 different strains;

-> Segmented influenza genome gets reassorted (e.g the new virus contains 3 segments of strain X and 5 segments of strain Y)

• influenza clinic: cause of severe respiratory infections and hospitalization in old people

• natural reservoir of influenza A: poultry, pig as “mixing vessel”, spread into human population

• natural reservoir of influenza B: humans are the only hosts

• influenza: what does H1N1 or H5N1 mean? Serotypes are determined by H and N antigens. Different antigen-families exist that can be combined. HxNy indicates which antigen families are present in a particular strain

Viral persistence: The virus (its particles or genomes) remain in the body over a long time or lifelong

Latency: The virus persists but does not replicate. Latency is controlled at the genome level, e.g. circular viral DNA (Herpesviruses), integrated DNA (retroviruses)

Productive infection: viral replication is active, viral particles are being formed

Reactivation: switch from latency to productive infection

LATs: latency-associated transcripts

miRNA: micro RNA

• different types of infection patterns with viral examples:

• acute infection (influenza, SARS-CoV-2)

• chronic infection (HIV, HCV (80%), HBV (5%)

• latent infection with episodes of reactivation (herpesviruses)

• chronic and latent infections are persistent infections

• HSV subfamilies and their location of persistence:

• alpha-herpes in sensible neuroganglia

• beta-herpes in lymphocytes/monocytes, kidney, salivary glands

• gamma-herpesviruses in B lymphocytes

• disease symptoms caused by HSV-1/2: herpes labialis, herpes genitalis

• replication cycle of HSV-1: entry, concept of immediate early and early and late genes with ”back to the nucleus”, genome replication, packaging, egress

• moa (mechanism of action) of acyclovir: nucleoside analog lacking 3’-OH, prodrug, phosphorylated by alpha-herpes thymidine kinase (monophosphate), phosphorylated by cellular kinases (triphosphate), incorporation into herpes DNA during DNA replication, chain termination due to lack of 3’ OH

• mechanism of HSV-latency: retrograde transport in neurons, HSV DNA as episome in nucleus, LATs (latency-associated transcripts) are processed into miRNAs; miRNAs suppress transactivators ICP4 and ICP0, switch from immediate early to early genes not possible, virus stays in latency

• symptoms of EBV infection: infectious mononucleosis/kissing disease (Pfeiffersches Drüsenfieber): fever, swallen lymph nodes, sore throat, fatigue, skin rash

• main mechanism of herpes genome replication: rolling circle

Genotype: variants of a virus that are genetically similar; they cluster in a phylogenetic tree

Serotype: variants of a virus that can be controlled by the same immune response (scenario lifelong immunity: if there is only 1 serotype, you get the disease only 1x in your life. If there are 2 serotypes, you can get the disease 2x in your life)

• ”big 7” viral causes of Childhood Diseases:

• mumps (mumps virus)

• measles/Masern (measles virus),

• rubella/Röteln (rubella virus)

• chickenpox/Windpocken(VZV)

• exanthema subitum/Dreitagefieber (HHV6 and HHV7)

• erythema infectiosum/Ringelröteln (PB19)

• Hand-Foot-Mouth-disease/ Hand-Fuss-Mund-Krankheit (enterovirus group A)

• what defines a “childhood disease”: endemic, highly contagious, life-long-immunity

• dissemination of measles virus in the body: infection via tonsils and pharyngeal lymph nodes, systemic infection, infection of the lungs, release of virus via aerosols

• childhood diseases during pregnancy or birth: VZV, BP19, rubella can harm the unborn child

• symptoms of VZV: primary infect causes chickenpox, reactivation causes herpes Zoster (Gürtelrose)

• cell cycle: G1, S, G2; M

• tumor suppressor genes: p53 and Rb inhibit cell proliferation by controlling S phase

• symptoms: warts (HPV1 and other), condyloma (low risk types HPV 6 and 11 and other) and cervix carcinoma (high risk types HPV 16 and 18 and other)

• PPV infection and cervix carcinoma risk: viral persistence (10%) and cancer (0.8%)

• How can HPV cause benign tumors (warts): infection of epithelial cells, E6 E7 expression, proliferation only in higher layers

• How can HPV cause cancer & why is HPV integration is key to cancer: infection of epithelial cells and basal cells, E6 E7 expression/proliferation also in basal cells

• episomal genome with unstable mRNA for E6/7, little E6/7 is formed, moderate inhibition of p53/Rb => no oncogenesis

• integrated genome with stable mRNA for E6/7 due to lack of AUUUA, overexpression of E6/7, strong inhibition of p53/Rb => oncogenesis

• targets of E6: p53

• target of E7: Rb

• vaccine? high risk HPV infection can be prevented by vaccination; the vaccine does not protect against CC if infection is already persistent because the vaccine does not contain E6/7, no immunity against E6/7 is formed

• intrinsic immunity: immune response within a single cell, e.g. TLR

• innate immunity: complex immune response directed at specific structures, e.g. NK killing of missing self

• adaptive immunity: complex immune response directed at variable structures, e.g. T- and B-cell response

• how receptors from intrinsic immunity can recognize foreign structures: unique ligands or unique loci

• 3 TLRs important for antiviral defense and their ligands: TLR3 dsDNA, TLR7,8: ssRNA

• 3 examples for intrinsic defense mechanisms: antiviral state shuts down protein synthesis; tetherin attaches enveloped viruses upon exit; APOBEC3G causes G-> A hypermutation; epigenetic silencing shuts down viral gene expression; apoptosis kills infected cells

• TLR-signaling ultimately results in interferon-alpha production

• Interferon-alpha can trigger an antiviral state

• MHCI: presents antigens to CD8+ T cells; present on all nucleated cells of the body

• MHCII: presents antigens to CD4+ T cells; present only on antigen-presenting cells

• Antigen-presenting cells: dendritic cells, macrophages, B cell

how B cells are activated to produce antibodies

• A viral antigen is taken-up by naive B cell via the BCR

• The B cell migrates to the Lymph node LN and presents the antigen via MHCII to effector TH cells

• The TH cell recognizes antigens presented by MHCII on B-cells releases activating cytokines

• The B cell develops into a long-lived plasma B cell that initially produces IgM and later IgG

• Some of these activated B cells develop into long-lived memory B cells

• Upon a secondary contact with the same virus or a booster vaccination, these memory B cells can quickly develop into new plasma cells (no need for an additional costimulatory signal from T cells) and produce high-affinity IgG

how T cells are activated

• A viral antigen is taken-up by dendritic cells

• The DC presents parts of this antigen on MHCII and (by cross presentation) on MHCI

• The DC migrates to the Lymph node (LN) and presents the antigen to naïve T cells

• a) An antigen-specific naïve CD4+ T cell recognizes the antigen on MHCII and develops into an effector TH cell

• b) An antigen-specific naïve CD8+ T cell recognizes the antigen on MHCI and develops into an effector CTL cell

• The effector TH and CTL leave the LN and migrate to a site of inflammation in the body

• The CTL recognizes infected target cells by the antigen presented on MHCI and kills the cell

• The TH cell recognizes antigens presented by MHCII on antigen-presenting cells in the tissue (e.g. macrophages that have taken up killed infected cells) and releases activating cytokines

• Most of the effector CTL and TH cells die after 2-3 days by activation-induced apoptosis

• Some of these cells survive and become long-lived memory T cells

• Upon a secondary contact with the same virus, these memory cells can quickly develop into new effector cells

simplified course of an immune response

• Every body cell has an intrinsic immunity to viruses or bacteria (part of the innate immune defense)

• A viral infection is recognized by dendritic cells (DCs) or macrophages via TLRs (Tolllike receptors)

• Activation of the TLRs leads to the release of interferon-alpha (IFNa)

• IFNa prepares the surrounding cells for a viral infection ("antiviral state"). Among other things, preparations are made to switch off the cell's protein biosynthesis

• As soon as these cells are also infected, they switch off their protein biosynthesis

• This brings viral replication to a standstill

• In addition, apoptosis is triggered in the infected cells

• In addition to interferon-alpha, the virus-activated DCs also release TNF-a and IL-6. These cytokines trigger the production of prostaglandin-2 (PGE-2) in epithelial cells, for example,PGE-2 triggers fever in the hypothalamus

• Usually, a flu-like infection is over after 2-3 days

• The virus has been eliminated from the body, the release of PGE-2 decreases and we are healthy again

• Although we have now recovered, the immune system still processes the previous infection over the following weeks: adaptive immunity develops...

• different classes of antibodies: IgM, IgG, IgA, sIgA

• IgGs can label virus-infected cells for killing via ADCC, CDC and ADCP

• the crucial need for mucosal antigen delivery if an sIgA response is wanted: sIgA is produced locally at mucosal sites. Simulation of mucosal-associated lymphoid tissue (MALT) is required to trigger class switching to IgA and its secretion across mucosal surfaces

adjuvant = substance that activates natural immunity/DCs to trigger an immunogenic immune response

ADCC = antibody-dependent cellular cytotoxicity

CDC = complement-dependent cytotoxicity

ADCP = antibody-dependent cellular phagocytosis

3 main principles of vaccines: live, antigen, gene-based

their characteristics:

• live: good CTL response, good antibody response, cannot be used in pregnancy, can cause vaccine disease

• antigen: weak CTL response, good antibody response, can be used in pregnancy, cannot cause vaccine disease

• gene-based: good CTL response, good antibody response, can be used in pregnancy, cannot cause vaccine disease

Live vaccines: replication-competent, attenuated virus

vector vaccines: replication-incompetent, transgene viral vector encoding for antigen

RNA-vaccines: mRNA encoding for antigen with proinflammatory LNPs

dead vaccine: inactivated whole virus with adjuvants

protein vaccines: recombinant antigen with adjuvants

• what „neutralizing antibody” means: can bind to surface of pathogen and prevent receptor-binding

• how mRNA vaccines work: proinflammatory LNPs activate DCs, mRNA is taken up by cells, cells translate antigen, antigen is presented on MHCI and MHCII, stimulation of CD8-T cells and CD4-T cells and antibody response

• meaning of IgG4 for ADCC, CDC and ADCP: dominantly blocks these processes, confers immunological tolerance

• Prime = 1st vaccination, activating the adaptive immune response, relatively low levels of IgG, formation of memory cells

• Boost = 2nd (or more) vaccination, activation of memory cells, high levels of IgG

• passive: transfer of antibodies, no long-lasting immunity, no T-cell response

• active: delivery of antigen to the body, activation of adaptive immune response and memory, long-lasting

• serial passaging of wild-type virus on different cell cultures and culture conditions (e.g. temperature)

• Evolution of virus variants that are optimally adapted to cell culture

• Selection of strains that have lost abilities to optimally replicate in the human body

• Caution: It is possible that the attenuated vaccine regains pathogenicity in humans (e.g. live polio vaccine).

Long-term stability of mod mRNA versus short-term stability of LNPs causes antigen expression in the absence of DC activation, consequence is induction of immune tolerance and switch to IgG4

• DNA plasmid with S gene in E coli,

• purification of plasmid,

• excision of S gene with t/protomor,

• in vitro transcription of mRNA in the presence of 7Me-PsU,

• capping,

• purification of mRNA,

• packaging into LNPs (lipid-based nanoparticles)

Live attenuated polio virus used for vaccination campaign, evolution of attenuated strains by serial passage (transmission from vaccinees to unvaccinated), regaining of pathogenicity