S 1: What is a virus?

- Obligate intracellular parasites cannot reproduce outside their host cell,

meaning that the parasite's reproduction is entirely reliant on
intracellular resources
S 2: Discovery
- was as early in 18th century when virus was describes as agent that
causes disease
- Pasteur said “Every virus is a microbe”
- Real discovery of virus started in late 19th century -> it was possible to
create filters which would retain bacteria (agent of tobacco mosaic
disease passed through filters that retain bacteria) -> clear that the agent
was a virus, NOT bacteria
- First 2 virus discovered: yellow fever virus (1901), rabies virus (1903)
- 1915: bacteriophages were discovered
- 1933: Influenza virus
- 1939: it was clear that virus are not liquids but particles + first EM of
bacteriophage
S 3: Virus classification
- Depends on nature of nucleic acid (DNA/RNA)
- Symmetry of protein shell
- Presence or absence of lipid membrane (envelope)
- Dimensions of virus particle
➔ Classical hierarchical system!
S4-6
- Virus has 2 phases: infectious particle (infects host) and when it infects a
cell
- Viruses don´t think and are passive agents
- Viruses mutate
- Viruses are all around us (skin, mouth, etc.) -> we regularly eat and
breathe billions of virus particles
- We carry viral genomes as part of our own genetic material
- List of viruses which infect us on S. 6
- Once infected, we are infected for life!
S7
- We carry microbiome: microbiome consists of microbes that are both
helpful and potentially harmful
- Many viruses don´t do anything to us, but they can potentially
S 8: Causes of 2017 global deaths
- Leading causes: cardiovascular diseases and cancers > ca. 17 mio
- Now with covid-19 pandemic > 6.2 mio (higher number compared to
HIV)
- HIV: ca. 900k
- Hepatitis > 100k (Hepatitis C is now curable)
S 9: Viruses are small
- Can only see them in Electron microscope
- Herpesvirus: 200nm
- Poliovirus: 30nm
S 10: Important definitions
- A susceptible cell has a functional receptor for a given virus -> without
receptor virus can´t infect it
- A resistant cell has no receptor -> virus can´t infect that cell
- A permissive cell has the capacity to replicate virus -> it may or may not
be susceptible
- A susceptible AND permissive cell is the only cell that can take up a virus
particle and replicate it
S 11: Propagation of animal viruses
- Reason why we have to replicate viruses is not only to study, but to
generate vaccines (all influenza viruses are based on attenuated or dead
viruses)
- Propagation in chicken eggs: depending which virus you want to
propagate it can be injected in chorioallantoic membrane, amniotic
cavity, etc.
- Chorioallantoic membrane inoculation: Herpes simplex virus, Poxvirus,
Rous sarcoma virus
- Amniotic inoculation: Influenza virus, Mumps virus
- Yolk sac inoculation: Herpes simplex virus
- Allantoic inoculation: Influenza virus, Mumps virus, Newcastle disease
virus, Avian adenovirus
S 12: Plaque essay
 - How can we count viruses? With a plaque essay (developed 1952)
- One bacterium is infected with bacteriophage, bacteria lyses and phage
gets spread on a plate and infects neighboring bacteria -> plaque
➔ The assay relies on determining the number of plaque forming units
(pfu) created in a monolayer of virus-infected cells. Plaques form when
a virus-infected cell Iyses, leading to a subsequent cycle of infection and
lysis of neighboring cells
S 13: Physical measurements of virus particles/Diagnosis (Counting viral
particles)
- Hemagglutination: if virus is added to red blood cells, the red blood cells
clot together -> from this clotting we can see how many viruses we have
- Electron microscopy
- Some viruses have special enzymes like RNA viruses have reverse
transcriptase and if we have an assay to monitor the enzyme activity, we
can see how many viruses we have
- Serology: based on antibodies, which can capture virus and we have 2nd
antibody which is attached to a protease -> enzymatic essay
➔ An antibody test (serology) looks for the presence of antibodies, which
are specific proteins made in response to infections. Antibodies are
typically detected in the blood of people who are tested after infection;
they show an immune response to the infection
- (RT-PCR is also another method)
S 14: Virus-antigen Detection
- This is an antigen test for sars-cov2
➔ Sample is applied (capillary flow is added) -> 1st antibodies are labeled
with covid, and 2nd antibodies are specific e.g., for spike protein
(control line; these antibodies don´t detect spike protein) -> they
move, and antibodies recognize covid -> control line won´t bind to
covid
- mABs – monoclonal antibody
➔ Während des Tests werden Probenextrakte auf die Testkarten
aufgebracht. Wenn der Extrakt SARS-CoV-2- Antigen enthält, bindet
das Antigen an den monoklonalen SARS-CoV-2-Antikörper. Während
des lateralen Flusses bewegt sich der Komplex entlang der Membran
zum Ende des absorbierenden Papiers. Beim Passieren der Testlinie
(Linie T, beschichtet mit einem anderen monoklonalen SARS-CoV-2-
Antikörper) wird der Komplex von SARS-CoV-2 eingefangen
S 15
- Cycle Threshold (CT): can calculate virus load -> at the beginning, virus
load is not much, from CT value onwards we see exponential growth of
virus
- High throughput sequencing is used to detect new variants of sars cov2
S 16: Virus attachment and entry
- 1st stage is Attachment and Entry -> Transcription and Translation of
viral genome -> Genome Replication (DNA or RNA) -> Assembly -> last
stage is Release of virus
- Essential for all virus (except those of fungi and plants) is a receptor
- 1985: one receptor known: sialic acid for influenza virus
S 17: Criteria for identifying cell receptors for viruses
- Receptor binds virus particles
- Host cell can generate antibodies against receptor (blocks infection) e.g.,
if we vaccinate against spike protein of sars-cov2 it blocks receptor
binding protein
- Receptor gene confers susceptibility (more than one may be involved
and some cells have this receptor others don´t) -> this receptor allows
entry of virus
- Disruption of receptor gene blocks infection -> e.g., with crispr-cas9
(receptor doesn´t become susceptible anymore)
S 18: ACE2 is receptor for Sars-cov2
- HeLa cells aren´t susceptible to sars-cov2 because they don´t produce
ACE2
- Sars-Cov2 requires ACE2 (Angiotensin-converting enzyme 2) as receptor
- DAPI, FITC, Np-Cy3: fluorescent stains, which detect viral nucleus
S 19, 20: Viral receptors
- Viruses of the same family may bind different receptors (e.g., rhino-and
retrovirus)
- One virus may bind multiple receptors
a) HSV-1 binds heparan sulfate proteoglycan (HSPG), Integrin, Nectin-1,
herpesvirus entry mediator (HVEM), 3-OS-HS (3-O-Sulfated Heparan
Sulfate
b) Influenza virus binds different receptors of sialic acid (alpha (2,6) –
human strains; alpha (2,3) – avian strains)
S 21: Sars-cov2 Attachment
- ACE2 receptor is recognized by spike protein of virus
- Spike protein is a dimer, which undergoes conformational changes until
virus infects cell
- Conformational changes happen through cleavages by furin
- Cleaved at S1/S2
➔ Spike protein needs to be cleaved first, so that it can attach to a
human cell
S 22: Sars-cov2 entry
- Protease cleavage of spike protein needed for fusion: this process must
be regulated
➔ Antibodies for spike protein don´t recognize the final protein
because of conformational changes
S 23: Entry at plasma membrane
- HIV-1 uses its viral protein gp120 to bind receptor CD4 and co receptor
CCR5 -> conformational changes triggers uptake of virus
- Same with Paramyxovirus and its attachment protein
S 24: Endocytosis
- Uptake of virus through endocytosis
- Virus is first in endosome and DNA/RNA is replicated
- E.g., Adenovirus
S 25: Uncoating of adenovirus at nuclear pore complex
- Other viruses don´t have nuclear core like Adenovirus -> attached to
Dynein and Kinesin (along microtubule) -> specific proteins on nuclear
pore complex -> viral DNA gets into nucleus -> DNA can now go through
transcription
S 26
- Similar process with poliovirus and Vp1, Vp4
S 27: HIV Assembly and Release
- This virus has reverse transcriptase and RNA converts into DNA -> DNA
can persist in our genome longer
S 28ff: Acute infections
- Acute infection is a short but also severe course of disease
- E.g., influenza, polio, measles, norovirus, west Nile virus, sars-cov2
 - Course of a typical acute infection
a) At the Beginning (2 days) slow growth of virus
b) After few days virus starts replicating and adaptive immune system is
induced (antibodies, etc.)
c) Adaptive response
d) After virus is cleared -> lifelong Memory
- E.g., sars-cov2 (after infection of 1st variant -> memory), but with
mutations you can be infected again
S 29: Influenza
- Types A, B, C
- Only Types A, B cause disease
- C is mostly not apparent or very mild
- Only A causes pandemics
- USA 2019/20: 39-56 Mio flu illnesses; small percentage get to hospital
and only very few die
- In Austria: 1500-3k influenza deaths

S 30: Types A, B and C

- Changes in proteins cause new subtypes
- Newly generated every year
- Getting flu shot can prevent illness from types A and B
- Proteins HA and NA are antigens which means they are recognized by
immune system and trigger an immune response
S 31 - 34: Antigenic Drift and Shift + Influenza in animals
S 35: Influenza pandemics
- The most severe pandemic was 1918: Spanische Influenza (originally
from US, but first detected in spain) – end of 1st world war (worldwide:
50 Mio deaths) was caused by H1N1
- 2009: Schweinegrippe same strain as Spanische Influenza

S 36, 37: Norovirus

- Need only few particles (18) that cause disease
- Causes 50% of all food-borne outbreaks of gastroenteritis in US
- Diseases are very harsh
- Fecal-oral spread
 - Virus particles retain infectivity passing through stomach -> don´t get
killed by stomach acid (they are still infectious even after going through
stomach)
- Infect vili in gut (infect cells in vili: intestinal epithelial cells (IEC),
macrophages, dendritic cells and T cells)
- Basis for vomiting, diarrhea not known
S 39 – 41: West Nile virus
- Was detected in 1937 in west Nile district of Uganda
- Absent from western hemisphere until 1999
- Somebody infected brought it by plane to US -> since then cases in NY
- 2002 spread almost over US, 2006 all of US
- Transmitted via Culex pipens (common mosquito) which infect humans
and other hosts like horses
- WNV pathogenesis! 1 out of 150 individuals develop neuroinvasive
disease
S 42: Hepatitis virus
- Causes acute disease
- Vaccination for Hep. A and B
- Hep. C was problem for long time, because in Africa, S-America, and Asia
there are 170 Mio cases, and it can cause liver cancer
- Now there is medication for Hep. C which is based on inhibitors of
proteases which are required to cut polyprotein of virus which is
translated -> Hep. C virus has its own RNA polymerase, and this
polymerase is inhibited
- But medication is expensive (except Italy where medications aren´t
made too expensive)
S 43, 44: Origin of Sars-cov2
- Closed Borders in Canada -> few cases
- Sars-cov1 had much more severe outcome than sars-cov2
- > 60 years mostly already have coexisting illness
- Dyspnea = shortness of breath
- ICU = Intensive care unit

S 45, 46
- Most prominent protein, spike protein, binds to receptor and other
prominent protein is nucleocapsid protein
- Replication cycle on p. 46
 - Can infect cell by fusion or endocytosis -> RNA replication -> structural
proteins go to endoplasmatic reticulum and a nucleocapsid is made in
cyctoplasm of cell -> exocytosis
S 47ff: Persistent infections
- Virus is always there, may or may not have an impact
- E.g., HIV-1
S 48, 49: HIV-1
- Is a lentivirus
- First isolated in 1983
- 1984: blood test developed
- 1921: virus jumped from monkey to human -> 75 Mio Infections; 32 Mio
Deaths
- There was denial that HIV is infectious agent of AIDS
- AIDS occurs with the Kaposi´s sarcoma -> skin cancer (described in 1872)
- Occurs in HIV affected people because immune system gets weaker and
chance that you develop skin cancer is higher
- Co-infection with human herpesvirus 8 is necessary for development of
Kaposi´s sarcoma
- AIDS denialists: the hypothesis that HIV-1 causes AIDS has been tested
by inadvertent/accidental infection of people with HIV-1 contaminated
blood
S 50: HIV-2
- Less virulent than HIV-1
- No mother-infant spread
- Spread in Africa
- Crossover from monkey sooty mangabey to humans
- 8 distinct lineage with different variants

S 51
- 2018: 37.9 Mio people living with HIV
- Austria: 40k active cases
- Why are the cases getting more and more ? People are less careful, and
cases increase because People don´t die as much as before -> the longer
you live, the higher is chance to transmit virus (especially without
symptoms)
S 52: Control of AIDS
 ➔ As the HIV epidemic matures, increasing numbers of people are
reaching advanced stages of HIV infection. Antiretroviral therapy
(ART) has been shown to reduce mortality among those infected and
efforts are being made to make it more affordable within low- and
middle-income countries.
- No cure, no vaccine
- People can´t stop taking antiviral drugs
- Problem by antiviral drugs: Drug resistant viruses appear
- Drugs are expensive
S 53: HIV-1 diversity
- 4 groups based on sequence alignment (envelope region)
- M, O, N, P
S 54: Co-receptors + susceptibility
- HIV binds to CD4+ T cells receptors
- HIV uses CCR5 receptor or CXCR4 receptor (targets CD4 cells and
macrophages)
- 4-16 % of all Europeans have mutation in CCR5 receptor which protects
from HIV-1 infection -> but with this mutation you are highly susceptible
to West Nile virus
S 55: Progression of HIV infection
- Acute phase with not that severe symptoms (fever, diarrhea)
- Chronic phase (8-10 years) with no symptoms
- Symptomatic phase (after 8 years) -> development of AIDS and therapy
is needed + susceptible to other diseases with weakened immune system

S 56: HIV and cancer

- Cancer develops in 40% of infected individuals
- High levels of cytokines lead to uncontrolled cell proliferation and
replication of oncogenic viruses
- Endothelial cells aren´t controlled and B cell will turn into B cell
lymphoma caused by cytokines
- Epithelial cells will proliferate into carcinoma
- Endothelial cells -> Kaposi´s sarcoma
➔ B cell lymphoma: A type of cancer that forms in B cells
➔ Kaposi's sarcoma (KS) is a vascular tumor of proliferative endothelial
cells
 ➔ Carcinoma is the name given to a group of cancers that start in
epithelial cells
S 57ff: Vaccines and Antivirals
- Vaccines are easiest way to control viruses
- There are active and passive vaccines
- Active: inject attenuated virus to a patient -> long term protection
- Passive: inject antibodies or immune cells (products of immune
response) to patient -> short term protection (works for acute
infections)
S 58: Active therapy
- 1796: Jenner injected Cow pox virus to prevent VCV infections (Varizella
Zoster virus)
- 1885: rabies vaccine from Pasteur (vacca in latin = cow, because cow pox
was 1st vaccine)
- 1930s: Yellow fever, Influenza vaccines
S 59: Passive therapy with
convalescent serum
- 1969: Jordi Casals infected himself with Lassa virus
- Transfused with blood from nurse who had survived Lassa fever
- Ongoing trials of convalescent plasma for Covid-19 patients
- Blood transfusions of people who have survived Covid-19
➔ convalescent plasma therapeutic approach is based on the principle
of passive antibody therapy, a short-term strategy whereby
antibodies from the blood of someone who recovered from an
infection can be administered to protect or treat another person.
➔ The end goal is the same as vaccines -> making antibodies against a
specific infectious agent readily available.
mAB
- antibodies are available against viruses
S 60: Active Immunization
- attenuated virus
- inactivated virus (heated, etc.) but there is problem
- fractioned virus (purified subunit vaccine)
- virus cloned into bacterial cell -> DNA/RNA vaccine or virus vector
vaccine or expressed as protein: subunit vaccine and virus like particle
vaccine
S 61ff: Inactivated vaccines
- through chemical procedures (e.g., formalin, beta-propiolactone, etc.)
which inactivate DNA or RNA -> no replication of virus anymore
- infectivity is eliminated, antigenicity not compromised
S 62: Inactivated Poliovirus vaccine, IPV
- treated with formalin to destroy infectivity
- 1954: produced in Institute in san diego
- Cutter incident: small company didn´t have virus completely inactivated
and people died from it after vaccination
S 63: Inactivated Influenza virus
- Envelope proteins change each year
S 65: Recombinant zoster vaccine – Shingrix
- surface protein produced in human cells
- This vaccine is based on glycoprotein E (gE) of varicella zoster virus
(VZV), the most abundantly expressed protein of VZV
- The recombinant gE of Shingrix is expressed in Chinese hamster ovary
(CHO) cells.
- problem with all (inactivated or subunit) vaccines is that they cause a
mild immune response -> adjuvants are needed
S 66: Adjuvants
- ASO1/4
- Alum
- MF59
S 67: Sars-cov2 Vaccines
- 11 in use!
S 68: Sars-cov2 Spike protein
S 69: Moderna mRNA-1273
- LNP (lipid nuclear protein) particles are being injected in muscle ->
immune cells move to the draining lymph node -> proteins are produced
-> LNP uptake and antigen expression in cells at the injection site and in
draining lymph nodes
S 70: Thoughts on Covid-19 vaccines
- T cells kill infected cells (more potent than developing IgG antibodies)
- Antibodies protect but there are not many
 - While T cells are everywhere and therefore development of T cell
epitopes for future vaccines is made
- 2 reasons why T cells are more beneficial:
a) Antibodies are not that many, while T cells are everywhere
b) New covid-19 virus variants have changes in antibody epitopes, while
most T cell epitopes are not changed
S 71: Vaccines can prevent viral disease
- But they have almost no therapeutic effect if an individual is already
infected!
- Therefore, we have second antiviral defense: Antivirals
- Antivirals can stop infection once it has started
S 72 - 73: Antiviral drugs by virus and target
- But antivirals have side effects
- Antivirals interfere with virus growth which can indirectly affect host cell
- Most important reasons:
a) Antiviral must completely block viral replication, if it doesn´t do this,
we get resistant mutants
b) Acute infections are of short duration:
By the time patient feels ill, it´s too late to impact disease with
antiviral drugs -> antiviral drugs for these viruses must be given early
in infection (safety issues: giving drugs to healthy people isn´t wise)
S 74: Significant hurdles stand in the way of finding effective antiviral drugs
- Antiviral drugs costs from $100-200 Mio
- Graph: 100k compounds (antiviral drugs) are synthesized or purified -> in
the next 10-15 years almost all of them get rejected because of many
reasons!
S 75: Antivirals target different steps during the viral replication
- Prevent attachment, uncoating, RNA/DNA synthesis, Polyprotein
processing, integration, assembly of virus
➔ All target HIV-1, except mRNA synthesis: Influenza virus
S 76: Entry Inhibitors
- Influenza virus: amantadine blocks entry of protons into virion and
prevents uncoating
- CCR5 inhibitor: Maraviroc
➔ Have to take drug before you get into contact with virus
 ➔ Therefore, entry inhibitors only good for people who are in danger of
getting in contact with virus -> usually don´t know that!
S 77 – 78: Polymerase Inhibitors
- Acyclovir inhibits herpes simplex virus
- AZT: 1st HIV-1 drug (blocks reverse Transcription – RT)
- But resistance through mutants to AZT happened (Altered RT don’t bind
phosphorylated AZT)
- Therefore, combination therapy of 2 antiviral drugs was introduced
S 79: Chemotherapeutic agents approved for HIV/AIDS treatment
- Nucleoside analogs, reverse transcription inhibitors, protease inhibitors
and fusion inhibitors
- HAART (high active anti-viral therapy)
➔ HAART steht für hochaktive antiretrovirale Therapie, im Englischen
"Highly Active Anti-Retroviral Therapy" = eingeführte
Kombinationstherapie, welche bei stattgefundener HIV-Infektion den
Ausbruch von AIDS verhindern soll.
➔ Im Rahmen der HAART wird eine Kombination aus drei (oder vier)
antiretroviralen Wirkstoffen eingesetzt, hierbei werden meist
folgende Wirkstoffklassen kombiniert: Zwei nukleosidische Reverse-
Transkriptase-Inhibitoren (NRTI) mit einem nicht-nukleosidischen
Reverse-Transkriptase-Hemmer (NNRTI) oder mit einem
Proteasehemmer, welcher durch eine geringe Dosis Ritonavir
geboostert wird oder mit einem Fusioninhibitor
S 80: Hep. C virus RNA polymerase inhibitors -> Sofosbuvir
S 81 – 82: Protease Inhibitors
- Ritonavir: HIV
- Telaprevir: Hep. C
S 83: Pfizer (combination drug: Paxlovid)
- Nirmatrelvir inhibits Sars-cov2 3C-like protease (3CLpro)
- Ritonavir inhibits cytochrome P450 and Cytochrome P450 inactivates
nirmatrelvir
- Taking Nirmatrelvir and Ritonavir at the same time, to slow down the
disruption of Nirmatrelvir in the body by cytochrome p450
S 84: Mathematics of drug resistance + Combination therapy
- Because of things mentioned here we need combination therapy -> one
therapy usually doesn’t work
- Not used for long term diseases like HIV, because doesn´t cure infection
-> latent reservoir remains