Virology Flashcards
give 6 distinguishing features of viruses
• small size (20-2300 nm)
• obligate intracellular parasites: need a host cell for replication
• simple composition: simplest have only protein and nucleic acid
• unique mode of replication (not binary fission)
• great diversity: infect all cellular organisms, cause devastating plagues or asymptomatic
infections
• numerous: estimated 1031 virions in biosphere
how did we originally discover viruses were v small
Viruses were discovered as disease-causing agents that passed through filters that
retained all bacteria, and hence were very small.
Give the range of virus sizes, including examples
The smallest, such as foot and mouse disease
virus (FMDV), are only 20 nm in diameter, whereas others, such as poxviruses, are bigger (250
x 350 nm), and the largest, the mimiviruses, are up to ~700 (diameter) and ~2300 nm
(length).
True or false
Viruses are always smaller than bacteria
false
the largest, the mimiviruses, are up to ~700 (diameter) and ~2300 nm
(length). These are larger than the smallest bacteria (micrococci ~ 500 nm).
What is the simplest viral particle
What is it essentially
the virion
a nucleic acid (genome)
surrounded by a protein shell (capsid) that protects the genome from the environment and
delivers the virus genome from one susceptible cell to another.
What are capsids composed of
repeating protein subunits (capsomers) that are arranged in a
symmetrical array. Symmetry is, in nearly all cases, helical or icosahedral.
The capsid of plant some viruses is made of many repeated identical polypeptides. What does this mean for the viral genome
only one gene is needed to make the capsid as it is just lots of the same gene product bound together
How do animal virus capsids differ from plant virus capsid
animal viruses are often more complicated - each subunit may be composed of several polypeptides and there may be more than one type of subunit.
True or false
All viruses all contain protein and nucleic acid
Some viruses have only protein and nucleic acid, but others contain lipid and carbohydrate too.
How are viruses which contain lipid and carbohydrate different in structure to those that are only nucleic acid and protein
in virus with carbohyrate and lipid, the capsid is surrounded by a phospholipid membrane (envelope) that is acquired from the host cell
How is the envelope of some viruses often made
What is it embedded with and what can happen
often by budding of the nucleocapsid through
the plasma membrane.
The membrane is embedded with viral proteins which, like many membrane proteins, may be glycosylated.
Define the following:
virion
virus genome
capsid
Virus particle = virion
Viral nucleic acid = genome
Protein coat = capsid
what are the following
capsid protein
capsid+genome
viral membrane
Capsid proteins = those proteins found in the capsid
Capsid + genome = nucleocapsid
Viral membrane = envelope
What are the following
envelope proteins
structural proteins
non structural proteins
Envelope proteins = viral proteins embedded in envelope
Structural proteins = proteins found in the virion
Non-structural protein = virus protein expressed in the infected cell but absent from the virion
Describe the viral genome generally
Virus genomes are composed of DNA or RNA, which may be linear or circular, monopartite
or segmented, and double stranded (ds) or single stranded (ss)
What are the 2 classes of ssRNA genome
If the RNA is mRNA sense (i.e. can be
translated into protein) it is a positive-strand RNA genome. If the genome is complementary
to mRNA (i.e. the mRNA is obtained by transcribing the virus genome as template), it is a
negative-strand RNA genome
Is it more common for a viral genome to be segmented
no
Usually, the genome is a single nucleic acid molecule (monopartite), but a few viruses contain
several nucleic acid molecules (segmented); example, influenza virus and rotavirus.
what is the size of viral genome limited by?
the error prone nature of RNA polymerases.
If the genome is too big (> ~20 kb) replication creates too many mutations, which are lethal
What is the usual RNA genome size for a virus
> 15kb
What sizes are the largest RNA viral genomes
What does this size necessitate
slightly over 30kb
proof reading activity of the RNA pol
Which viruses have the largest genomes
dsDNA viruses (mimiviruses: up to 1500 kb).
In the viral genome, how much does 1kb code for
If virus genomes range from 3kb to 1500kb, how many proteins are coded for at each extreme
1 mid sized protein
n (333
aa = 35 kDa),
the smallest genomes code for just a few proteins and the largest ~1500
Most viruses have small genomes and so limited coding capacity. Therefore, coding potential is
used efficiently by… (4)
- Densely packed genes
- Small intergenic spaces with few non-coding spaces
- Overlapping reading frames, use of a same nucleic acid to code for > 1 protein
- RNA splicing
How are viruses classified
grouped into families (viridae), subfamilies (virinae), genera, species and strains.
How were viruses classified in the past
Why can this cause confusion
different parameters such as the type of disease
caused, mode of transmission, capsid structure, immunological relatedness, genome
sequence, protein structure and mode of replication.
German measles vs measles: Rubella virus (a togavirus, +ve ssRNA, German measles) and measles virus (a paramyxovirus, - ve ssRNA) are viruses causing similar type of illness, but are caused by quite different viruses.
patients with Chicken pox and small pox look similar therefore they are caused by similar viruses. True or false?
false
caused by either a poxvirus
(variola virus) or herpesvirus (varicella-zoster virus), respectively.
What are arboviruses
The arboviruses are a large group of viruses that are transmitted by biting insects (ARthropod
BOrne VIRUSES).
What is classification of viruses now based upon
How are families arranged? why is this useful?
genetic relatedness and structural characteristic
into larger groups based on type of nucleic acid genome
groups viruses with similar replication strategies
Give 5 ways to study viruses
EM PCR haemagluttination immunological evidence of infection plaque assay
How are infectious virus titres expresses
How high can a titre go
plaque forming units per mL
some are a high as 10e9 or 10e10
how is EM useful for virology
what is it useful for
how is it done
enabled virus particles to be seen and their
structure studied.
This was useful for diagnosis and quantification.
E.g. by mixing a virus
preparation with a suspension of small beads of known concentration and counting both
virions and beads under the e.m., the concentration of virions may be deduced.
what does an em of a virus preparation measure
total virus particles, not how many are infectious.
How is PCR useful in virology
What is a possible draw back
genome sequences of many viruses are
known, so specific primers can be used in PCR to identify and quantify virus genomes.
This is very sensitive and useful for diagnosis.
But, like e.m., this does not measure virus
infectivity
What is haemagluttination
Some viruses bind to red blood cells (rbc) and cause their
agglutination (clumping), haemagglutination. So a crude measure of virus concentration
can be made by mixing virus dilutions with a standard number of rbc and determining
the maximum dilution of virus that can agglutinate the rbc. Note, this is not measuring
virus infectivity
Is haemagluttination measuring infectivity
NO
How does immunological evidence of infection help the study of viruses
What is it useful for
Infection will be followed by an adaptive
immune response, i.e. the presence of (or an increase in) antibody or T cell responses to
virus antigens.
This is usually retrospective and so seldom provides rapid diagnosis.
Useful for epidemiological studies.
Which measurement of viruses measures infectivity
plaque assay
How does plaque assay work
A dilution series of virus is applied to lawns of
susceptible cells. Where a virus binds to and enters a cell, it replicates and releases new
virions. These infect and replicate in adjacent cells. Eventually, a visible area of cells is
destroyed by the virus. This is a plaque. Each plaque derives from one infectious virus
particle, so the titre of infectious virus particles can be calculated.
infectious virus titre given in pfu/ml
Can the total number of virus particles and the pfu/ml differ?
yes
This ratio is called the particle / pfu ratio. For some
enveloped RNA viruses, the particle / pfu ratio may be >100 or >1000 to one.
can all viruses be titrated by plaque assay
no: hepatitis B cannot
true or false
there
is a phase during virus replication when an infected cell contains no infectious virus particles
true
What are the 3 phases of the virus life cycle
- adsorption and penetration
- eclipse phase
- assembly and release
How are the 3 phases of the virus life cycle illustrated
infecting cells with virus and measuring the virus pfu at various times thereafter
(the cells in each sample are lysed artificially to release intracellular virus).
Shortly after infection the virus titre drops to close to zero and rises again at the end of the eclipse phase.
The final titre is much higher than the input titre because the virus has multiplied.
Are the general features of the virus growth curve common to all viruses?
yes
but the timescale and virus yield vary greatly.
What is the latent period of the virus life cycle
give examples to demonstrate the difference between viruses
The time taken to form new particles
it is about 20 mins when
bacteriophage T4 infects E. coli, whereas herpes simplex virus takes about 10 hrs inhuman epithelial cells.
what is the mean burst size
The average yield of virus particles / cell
reflects the specific
virus - host cell combination, and is
influenced by the cell metabolic activity
Describe the binding of HIV
The HIV envelope protein, gp120 (glycoprotein of 120,000 Mr) binds to CD4 and a chemokine
receptor (the co-receptor). CD4 is limited to T-lymphocytes and macrophage/dendritic cells.
The tropism of HIV is defined by this
describe the binding of influenza
The influenza virus envelope glycoprotein (haemagglutinin, HA) binds to sialic acid (the
terminal sugar on most glycoproteins). Since sialic acid is on almost all cell surfaces, influenza
binds to most cell types and receptor-binding does not define influenza virus tropism.
What does EBV bind to on host cells
Epstein-Barr virus (EBV) glycoprotein 340 (gp340) binding to the cell surface protein CD21
What are neutralising antibodies in relation to virology
Antibodies against receptor binding proteins usually inactivate the virus by blocking infection
What must the virus do after binding
How can this happen
penetrate into the cytoplasm.
Penetration may occur at the cell
surface, or the virus may be taken into vesicles by endocytosis or macropinocytosis and enter
the cytoplasm after disruption of the vesicle membrane
How does penetration occur with enveloped viruses
fusion of the virus envelope and a cell
membrane at either the cell surface (plasma
membrane, neutral pH) or after endocytosis
with the endosomal membrane (at low pH).
What does the process of membrane fusion require
how is this acheived
2 membranes to be drawn close together followed by membrane disruption
conformational change in the receptor binding protein
Describe the penetration of HIV after binding
binding of gp120 to CD4 results in a conformational change in the virus gp120/gp41
and the virus envelope fuses with the plasma membrane at the cell surface
What happens to influenza after binding to sialic acid
the virion is
endocytosed and the endosome is acidified. The reduction in pH causes a conformational
change in the HA drawing the viral envelope close to the vesicle membrane. A hydrophobic
fusion peptide in HA is inserted into the vesicle membrane thus promoting fusion.
Why is hemagglutination not a problem for patients suffering from influenza
Influenza virus-induced ‘haemagglutinin’ is an ‘in vitro’ phenomenon that is useful for titrating influenza virus, but
has no in vivo significance.
What is the process of membrane penetration of non enveloped proteins
Binding of virus to receptor
• Conformational change of virus causing disruption of host membrane enabling
• Transfer of virus nucleic acid or entire capsid into cell
How does bacteriophage T4 penetrate the membrane
injects its DNA into E. coli by contraction of a syringe-like sheath
Why is it called the eclipse phase
What is happening
no infectious virus particles are present in the host cell.
The virus particle has been disassembled (‘uncoated’), the virus genome is being replicated and
virus proteins are being synthesised
What kind of control over virus protein synthesis is there
both temporal and quantitative
How is virus nucleic acid synthesis so regulated
Nucleic acid polymerases recognise specific sequence/structures (origins of replication).
Where do viral polymerases come from
Viruses must either provide their own
polymerases, or synthesise proteins that modify and exploit host polymerases.
What is the minimum requirement for a virus to code for
i) a virus-specific nucleic acid
polymerase (or evolve to utilise one from the host) and ii) for virus coat proteins.
What is a unifying theme for all virus despite their wide range of genome types and replication strategies
What does this mean
the need to translate their mRNAs on host cell ribosomes.
So if viruses are grouped according
to how they convert their genome into mRNA, viruses with common replicative strategies group
together.
Which viruses’ genomes cannot be translated and the host cell cannot transcribe them
How is this circumvented
negative strand ssRNA viruses (eg measles and rabies)
ds RNA viruses (rotavirus)
must use virus genomes must be transcribed into mRNAs by a RNA-dependent RNA polymerase encoded by the virus.
What does viral mRNA from negative sense ssRNA viruses act as
positive sense RNAs serve as mRNA and as a template from which more virus RNA (-ve sense) can be produced for packaging into new virions
True or false
purified virus RNA is NOT infectious
true
Where do most negative strand ssRNA viruses and dsRNA viruses replicate
Give an exception
Most of these viruses replicate in the cytoplasm, but influenza virus replicates in the nucleus.
Influenza is unusual in that it replicates in the nucleus. Why else is it unusual
it requires host DNA-dependent RNA polymerase II as well as the virus RNA polymerase to make virus mRNA
Name 6 positive strand ssRNNA viruses
poliovirus, foot and mouth disease virus, hepatitis A virus, rubella virus, yellow fever virus, chikungunya virus
What is the first step once a positive strand ssRNA virus enters the cell
genome is mRNA so can be directly translated
When the virus genome is mRNA the first step is translation. How does the virus replicate
The translated proteins include the RNA-dependent RNA polymerase that replicates the input virus genome via a complementary (-ve sense) RNA. This is then copied into more +ve RNA, which can be translated into more proteins or packaged into new virions
True or false
purified viral RNA is NEVER infectious
false
Purified viral RNA is infectious if injected into a susceptible cell.
What are retroviruses
+ve ssRNA viruses
but after entry the virus genome is
not translated.
retroviruses are not translated directly after entry into the cell. Why
Instead it is copied by reverse transcriptase (RNA dependent DNA polymerase) into a dsDNA intermediate that is integrated into the host genome (the provirus) .
Where is the reverse transcriptase found for retroviruses
within the virus particle
What is the strategy of the retrovirus after the action of the reverse transcriptase
The virus now has the strategy of a
DNA virus in that mRNA is transcribed by host DNA dependent RNA polymerase II
from the integrated provirus. The full length transcripts are either translated or packaged into new virus capsids within the cytoplasm. Some of the full length transcripts
need to be spliced to express some of the virus proteins.
Name 3 dsDNA viruses
adenoviruses, herpesviruses, papillomaviruses
Where do most DNA viruses replicate?
What does it use
what does this mean it will do as soon as it enters the cell
Most DNA viruses replicate in the
nucleus
use cellular machinery for the transcription,
transport and processing of
mRNA.
So after infection the virus
genome is transported into the
nucleus.
What does transcription of dsDNA virus genome use
Where is the mRNA translated
Where do the translated viral proteins go and what do they do here
DNA dependent RNA polymerase
II.
mRNAs are translated in the
cytoplasm
some of the proteins (DNA polymerase and capsid proteins) are transported back to the nucleus, where viral DNA is replicated and progeny genomes are package into new capsids.
Using your knowledge of dsDNA virus strategy and replication, tell me whether viral dsDNA is infection alone
it is infectious
the input virus particle has no nucleic acid polymerase. If virus DNA is purified and
injected into the nucleus, virus replication will occur. The viral DNA alone is infectious.
Why are poxviruses an odd type of dsDNA virus
replicate in the cytoplasm
Poxviruses also have dsDNA genomes but replicate in the cytoplasm. How is this possible?
What does this mean for the infectivity of the purified viral DNA
encode enzymes required for transcription of the DNA genome (DNAdependent RNA polymerase, and capping and polyadenylating enzymes) and package these
within the virion. The virus DNA-dependent RNA polymerase is needed for transcription from
virus promoters, so purified viral DNA is not infectious.
In general which viral proteins are needed early and late
, virus enzymes for genome replication (such as polymerases), or
proteins that modify the host cell, are required early. In contrast, capsid proteins that are used
to build new virus particles are required late.
Name 2 types of virus that have very particular control over temporal protein expression
Both herpesviruses and poxviruses have several temporally distinct gene classes that are expressed in a regulated cascade, with expression of the next class of gene being dependent on proteins of the previous class. These have been referred to as immediate early, delayed early and late (herpesviruses) or early 1, early 2, intermediate and late (poxviruses).
How does the quantity of expressed protein differ for viruses
Early proteins have regulatory or enzymatic functions and so are needed in low amounts. Late
proteins are mostly constituents of new virus particles and so are needed in large amounts.
Why is eukaryotic mRNA monocistronic
How is this a problem for some viruses (use an example)
How is this overcome
unlike
prokaryotic ribosomes, do not re-initiate translation after a termination codon.
poliovirus must encode all virus proteins
Translate the mRNA into a single giant polypeptide (a 'polyprotein') that is post-translationally cleaved by proteases into several, smaller, mature polypeptides.
Which viruses use polyprotein processing to produce their capsid proteins from a polyprotein
are the proteases that perform this cleavage from the host or virus
retroviruses
virally encoded so are potential targets for chemotherapy
What is an alternative to a virus having a single mRNA encoding all the proteins?
eg
have a genome with several
different RNA segments, each encoding a different polypeptide (e.g., influenza).
How do retroviruses place the coding region for some proteins at the 5’end
How does HIV do this
How is HIV pol made
splicing
The HIV envelope
protein env is made this way and
then cleaved into gp120 and gp41.
Pol is made as a polyprotein by
ribosomal frameshifting.
What happens after the eclipse phase
which enzymes are always required here
new virus genomes and proteins assemble to form new virions.
“self-assembly” can occur, i.e. no catalytic process is involved.
what does the formation of the helical rod of tobacco mosaic virus involve during assembly
progressive addition of protein subunits around the nucleic acid
molecule
how is a complete nucleocapsid formed from an icosahedral
an icosahedral structure may form from protein subunits alone and the nucleic
acid is then inserted into these ‘empty’ capsids to form the complete nucleocapsid.
Why may a virus only become infectious during assembly
Sometimes production of infectious virions is associated with the cleavage of capsid proteins
into mature forms, a step that is necessary for the virion to become infectious, e.g. HIV.
How is the release of progeny most simply achieved
how can this be achieved
what must the virus be careful to do
cell lysis
Some bacteriophages encode a
protein that causes lysis of the bacterium.
This must only be expressed late during infection
how do many enveloped virus progeny leave the cell
‘bud’ out of the
plasma membrane over prolonged periods, acquiring their envelope in the process.
Is virus replication the only outcome of infection?
no
Some viruses can infect a cell and remain in a
quiescent state within the cell. This is called latent infection
What happens in a latently infected cell
contains
the viral genome but no virus multiplication occurs, yet the cell has the potential to produce
progeny virus - i.e., to switch from latency to the productive cycle.
Name 2 virus types that can become latent
retroviruses and herpesviruses
What happens after the retrovirus infects a cell broadly
When would it become latent
viral genome is converted to dsDNA by reverse transcriptase and then DNA integrates into a host chromosome to form the provirus
if the proviral DNA is not transcribed so no viral proteins are made
Why can it be hard for the body to eliminate all HIV infected cells
latent cells do not have any viral proteins so cannot be detected by the immune system
How can retroviruses be transferred vertically
if the virus integrates into germ cells
Give evidence that our evolution was driven by retroviruses and retrotransposons
The human genome is riddled with bits of retroviruses and retrotransposons that represent 8%
of our DNA.
In contrast, only 2% of our DNA codes for proteins
How do herpes viruses become latent in infected cells
they are dsDNA so must head straight to the nucleus to be transcribed.
but in some cell types there is no
transcription (or very limited transcription) of the viral genome. The viral DNA is quiescent -
does not integrate (with rare exception) but exists as an extrachromosomal
circular molecule called an episome)
What does the latent viral DNA in a cell infected with herpes exist as
as an extrachromosomal
circular molecule called an episome
How does a latent infection switch to a productive cycle
What is this called
might
occur when changes in the transcription factors in the cell allow recognition of viral promoters.
reactivation
give 2 examples of latent herpes infection
• herpes simplex virus (HSV-1) causes repeated cold sores
• varicella-zoster virus (VZV) primary infection causes chicken pox, reactivation
causes shingles
Virus infection of a cell is more than just replication followed by release. Give 8 ways in which the virus can modify an infected cell.
• Subversion of cellular metabolism to make only viral macromolecules
• Stimulation of cell biochemistry to enhance yields of virus
• Expression of virus enzymes to enhance nucleoside triphosphate (NTP) pools and so
increase nucleic acid synthesis
• Cell membrane modifications
• Induction of morphological changes to the cell (cytopathic effect, cpe)
• Evasion of host sensing of infection - blocking activation of innate immunity
• Non-lytic infection – persistent or latent infection
• Cell transformation - cancer
Describe how poliovirus subverts cellular metabolism
Within 1 h of infection the infected cell stops making host proteins and only makes poliovirus proteins. The virus has turned the cell into a virus factory whose only purpose is poliovirus replication
How does poliovirus stop host ribosomes recognising host mRNA
viral protease cleaves 5’ cap binding complex so it can longer recognise 5’cap
How is poliovirus mRNA translated
host ribosome recognises viral IRES after 5’cap was cleaved
Name 3 ways the virus can shut off translation of host proteins
cleavage of 5’cap recognition complex
destruction of host DNA
destruction of host mRNA
How do poxviruses subvert cellular metabolism
encode enzymes that cleave off 5’-caps from virus and cellular
mRNAs. Virus mRNAs are much more abundant and so predominant. Therefore, the translated
proteins are viral
What is a benefit of the poxvirus method of subverting host cell metabolism
helps the virus switch from early to late virus gene
expression more rapidly because the early mRNAs are destroyed once their synthesis stops.
Why would a virus want to stimulate the cell cycle
DNA viruses require high levels of dNTPs, but in resting cells dNTP
pools are low. Non-replicating cells (i.e., most cells) are therefore poor hosts for DNA viruses
Having stimulated the cell, viral DNA
synthesis and capsid protein synthesis occur more efficiently.
How can a virus stimulate cell cycle
Some DNA viruses (papovaviruses, adenoviruses,
herpesviruses) synthesise factors that stimulate the cell into cycle (e.g simian virus 40 T
antigen) . These factors are made early in infection
Vaccinia virus expresses an epidermal growth factor that is secreted from the infected cell and stimulates neighbouring cells to divide, making them
ideal infection targets.
How do pox viruses stimulate cell cycle
Vaccinia virus expresses an epidermal growth factor that is secreted from the infected cell and stimulates neighbouring cells to divide, making them ideal infection targets.
Name a factor synthesised in a virally infected cell that stimulates cell cycle
simian virus 40 T antigen
Is stimulating cell cycle the only way to increase the virally infected cell’s pool of dNTPs
no
the virus to express its own enzymes
that produce dNTPs.
Why does poxvirus and herpes encode for thymidine kinase
Larger DNA viruses (poxviruses and herpesviruses) encode enzymes that produce dNTPs: for instance, thymidine kinase, thymidylate kinase and ribonucleotide reductase.
When might a poxvirus growth well in actively growing cells in culture but poorly in resting cells
How would they affect an animal
if they are lacking the genes for dNTP producing enzymes
cause no disease (are avirulent) in animals.
What are non essential genes in the viral genome
eg genes that code for dNTP producing enzymes
THIS DOES NOT MEAN THEY ARE NOT IMPORTANT IN VIVO - it just means they are unlike genes encoding capsid proteins or viral polymerases,
which are essential
Why do enveloped viruses modify the cell membrane
part of their replication cycle, because the plasma membrane
will become the virus envelope and must contain the proteins required for adsorption to and penetration of the next host cell.
What can the effect of viral modification of infected cell membrane be (2)
may be to change
the behaviour of a cell with respect to its neighbours,
make it a target for NK cell
recognition
How does measles affect the host membrane
s induces the infected cell to fuse with surrounding uninfected cells so spreading
virus to uninfected cells without exposure outside the cell (and a target for antibody)
What are cell-associated viruses
Name one
Viruses
that pass from cell to cell without an extracellular stage
Measles
What is cpe
cytopathic effect - when a virus-infected cell shows a strikingly different morphology to uninfected cells
What can cpe be caused by
e alterations to the cytoskeleton (actin and tubulin containing filaments), which are exploited by the virus to facilitate intracellular movement of virus particles.
Give 3 viruses that can be identified from the cpe they result in
Rabies virus: Negri bodies in Purkinje cells in cerebellum
• Human cytomegalovirus: nuclear inclusion bodies that resemble “owl eyes”
• Poxviruses: cytoplasmic, eosinophilic inclusion bodies
How do cells sense viral infection
what is the effect on the virus
Cells sense virus infection by detecting the presence of (PAMPs), such as virus nucleic acid, by pattern recognition receptors (PRRs).
leads to an innate immune response, which left unchecked are detrimental virus replication and spread
Give an example of how viruses mitigate the effect of the innate immune response
Large DNA viruses, such as herpesviruses and poxviruses, encode many proteins that block the innate immune response to infection
What are 3 rules to remember when considering if a viral infection is lytic or non lytic
The productive cycle of DNA viruses is lytic.
Non-enveloped RNA viruses are lytic.
Viruses that cause host shut-off are lytic.
Non-enveloped RNA viruses are lytic. What about enveloped RNA viruses?
some enveloped RNA viruses, including retroviruses, can multiply in cells without killing
them and so release virus particles over a long period of time.
What does it mean to say a virus transforms a cell
the
cell now exhibits uncontrolled growth, fails to respond to the presence of neighbouring cells
(contact inhibition), and has many of the properties associated with malignant cells in vivo
What amount of human cancers are caused by viruses
20%
How do certain DNA viruses transform the cell
stimulate cell metabolism to create a suitable environment for virus
replication. This stimulation (by an ‘early’ virus protein) is normally followed by virus DNA
synthesis, virus capsid protein synthesis, the appearance of new particles and cell death
Give an example of a DNA virus transforming a cell
What can go wrong
papilloma viruses, which induce cell proliferation (resulting in a wart)
before synthesis of new virus takes place.
However, occasionally the virus replication cycle fails
(i.e. DNA synthesis and capsid protein synthesis do not occur) but ‘stimulation’ continues and
the cell continues to divide.
In the case of certain human papilloma viruses (HPVs) (notably HPV strains 16 and 18) cervical carcinoma may result.
How can retroviruses induce transformation
capture of oncogenes
integration to dysregulate cell division
How does retrovirus capture of oncogenes occur
Retroviruses can occasionally acquire a host cell gene during replication. If the cellular gene
plays an important role in the control of cell growth, then the resultant virus will ‘transform’
cells because the gene will be expressed at abnormally high levels, and lacks normal regulation,
when the virus infects a cell.
What is Rous sarcoma virus
an acute transforming retrovirus
it is an avian retrovirus that acquired src gene leading to src RTK being overexpressed
Why are acute transforming viruses usually replicative defective
What do they now require
Usually the acquisition of host gene is accompanied by loss of virus sequences.
co-infection by a helper virus to replicate
What makes Rous sarcoma virus replication defective
lacks the envelope
gene (env) and cannot replicate unless a helper virus provides this
How can a retrovirus lead to cancer other than acquisition of an oncogene
random integration may disrupt a tumour suppressor gene or activate/overexpress an oncogene
this is rare
how commonly do retroviruses, other than acute transforming viruses, lead to transformation
very rarely
True or false
transformation occurs frequently in most viral infections
false
cell transformation by viruses is a rare event because it requires an abortive infection
(DNA viruses) or the activation of a key host gene (retroviruses)
more likely to occur when the virus persists in an individual for a long time
Name 5 virus types that are associated with neoplasia
some herpesviruses (EBV & HHV-8),
papillomaviruses (HPV 16 and HPV 18),
retroviruses (HTLV-1),
hepadnaviruses (HBV)
flavivirus (HCV
What must a virus do to survive a multicellular host
gain entry to the host, replicate despite the host defences, and be released in a manner enabling transmission to new hosts.
What are ‘portals’ in virology
Viruses generally enter and leave a host by a specific route (portals of entry and exit) and
these are closely linked to transmission between hosts
Do viruses have to enter and leave through the same tissue
All superficial (local) infections must use the same entry and exit tissue
Some systemic infections also use the same entry and exit routes.
What are the physical barriers that a virus must evade to gain entry (5)
the skin,
the cilia that beat on our mucosal surfaces to sweep foreign particles such as viruses and bacteria out of the respiratory system,
the mucous secretions that bathe these surfaces,
the proteases of the stomach and intestine
the low pH of the
stomach.
How do viruses overcome the physical barriers to their entry
eg?
they are specially adapted
viruses that enter via
the alimentary canal, such as poliovirus, must be highly resistant to the acid pH of the
stomach and the proteases that are present here and within the intestine. If other viruses that
do not usually enter via this route are ingested they would be destroyed quickly.
How are viral infections sensed by the innate immune system
nucleic acids that may either be in an unusual place (DNA in the
cytoplasm) or have an unusual structure (RNA with a 5’ triphosphate) and these are sensed as
foreign by PRRs.
Once viral nucleic acids are detected by PRRs, what happens
activate signalling cascades that
culminate in activation of transcription factors, eg NF-κB or IRFs.
Once activated, these transcription factors translocate into the nucleus where they promote
transcription of a wide range of genes that activate innate immunity. These include:
interferons, chemokines (that recruit leukocytes to the site of infection) and cytokines that
promote the inflammatory response (such as IL-1β and TNF).
What are interferons in relation to viruses
secreted glycoproteins that bind to specific receptors on cells to induce
activation of an anti-viral state. Subsequently, if a virus enters an IFN-treated cell it will be
unable to replicate
Give 2 ways to illustrate that IFNs are v important anti-virus chemicals
the much greater sensitivity to virus infection in the absence of IFNs or their receptors,
• the fact that very many viruses, probably all mammalian viruses, have at least one way of
avoiding or disabling IFNs, or the functions of IFN-induced proteins
How can a virus be detected once it has infected a cell, if not by a PRR
TLRs on the endosomes that contain the virus
Give the cascade following detection of a virus by a PRR
PRR activates IRF3 and NF-κB, which activate IFN beta transcription
IFNβ is secreted from the cell where it binds to the type I IFN-R on adjacent cells.
activates JAK-STAT pathway, activating ISGF-3
ISGF-3 binds to ISRE and ISGs are activated, rendering the cell resistant to viral infection
What are the following
a) ISGF-3
b) ISRE
c) ISG
a) a transcription factor complex interferon stimulated gene factor 3 (activated by JAK-STAT pathway)
b) interferon stimulated response element, which ISGF-3 binds to
c) interferon stimulated genes, whose protein product leads to priming of the cell to be resistant to viral infection
give 3 examples of ISGs
protein kinase R (PKR),
2’-5’ oligoadenylate synthetase (OAS)
the Mx protein.
What do PKR and OAS require for activation
What do they do
what are the consequences (2)
dsRNA
(produced during infection by both DNA and RNA viruses)
inhibit protein synthesis (host and viral)
no virus
replication, and cell death.
Give 4 ways viruses can interfere with the interferon
Do any viruses exploit ALL of the these strategies
• stopping activation of the PRR-induced signalling cascades, so IFNβ is not produced
• releasing soluble proteins to bind IFNs and stop IFNs binding to the IFN-Rs on cells
• inhibiting the JAK-STAT signalling cascade to block induction of ISGs
• targetting the ISG proteins directly to block their action (e.g. 2’5’-oligo adenylate
synthetase, and protein kinase R)
yes poxviruses
Programmed cell death of a virally infected cell can be an effective way to limit spread of infection. How do viruses block apoptosis?
by blocking the action of
caspases (needed for induction of apoptosis), or targetting Bcl-2 family pro-apoptotic
proteins, which function at the mitochondrion to induce apoptosis.
What are chemokines
small chemo-attractant cytokines that recruit leukocytes to the site of infection. The recruited leukocytes then produce more IFNs or cytokines to activate T cells and
macrophages to fight the infection
why are inflammatory cytokines important during the response to viral infection
they drive the development of cellular immunity, such as CD8+ cytotoxic T lymphocytes
(CTL) that recognise and remove virus-infected cells. CTL are particularly important for
recovery from systemic virus infections.
How do herpesvirus and poxviruses block cytokine and chemokine action
secrete proteins from the cell that bind these molecules
outside the cell and stop them reaching their natural receptors
How does EBV block cytokine response
Epstein-Barr virus, expresses a viral cytokine (vIL-10)
that drives the immune system towards a Th2, rather than Th1 response.
How do NK cells kill virally infected cells
an antigen-independent manner.
NK cells kill virally infected cells in an antigen independent manner. what does this mean?
Normally cells express class I MHC molecules and the presence of class I MHC instructs the NK
cell not to lyse the cell. However, if class I MHC molecules are low or missing, as can happen
during virus infection, the NK cell is activated and kills the cell. Thus the cell may be destroyed
before virus replication is complete.
How is the killing of virally infected cells by NK cells different from CD8+ CTLs?
different activating stimuli:
For CD8+ CTL, the target cell is identified by the presence of specific virus peptides associated with class I MHC molecules on the cell surface, and this is antigen-dependent.
• In contrast, NK cells recognise the absence of class I MHC - may be antigen independent
NK cells are important early after infection before an antigen-specific CD8+ T-cell response
develops.
True or false:
NK cells can also develop pathogen-specific memory
true
What is the role of antibodies in a viral infection
how do they do this
Abs help prevent infection or diminish spread by free virions after an infection has
been established
bind to and neutralise virus particles, either alone or in combination with
complement.
What is mucosal IgA important for
preventing infection by viruses that enter by the
respiratory system
True or false
Abs are vital for removal of virally infected cells
false
more important in preventing infection or spread by free virions
What is the role of cytotoxic T cells in viral infections
CTL are unable to combat free virus particles, but are efficient at recognising and
destroying virus-infected cells
What kind of viruses are CTLs important in combating
viruses that remain largely cell associated (e.g. HCMV and measles virus) and for those that cause systemic infections.
Name a virus that is well adapted to block the CTL response
How
herpesviruses
have many strategies to block the presentation of peptides on class I MHC molecules
Herpesviruses have many strategies to block the presentation of peptides on class I MHC molecules. Give 4 examples
• Block generation of peptides by the proteasome
• Block transport of peptides into endoplasmic reticulum (HSV and adenovirus)
• Destroy class I MHC molecules by inducing their transport back into the cytosol for
proteolytic degradation (HCMV)
• Retain class I MHC molecules intracellularly and so preventing their transport to the cell
surface (adenovirus, VZV and HCMV)
Give 3 ways for viruses to escape adaptive immunity
• Latency. Hide from the immune system.
• Express Fc receptors on cells and virions. The Fc region of antibodies that are bound to
virus antigens on cells or virions is then not available to bind host Fc receptors.
• Antigenic variation: influenza, HIV and hepatitis C virus.
What are the 4 outcomes after a cell is infected by a virus
latency
cell death
persistent infection
cell transformation
Which cell types can poliovirus destroy?
What must you remember about this?
motor anterior horn cells in the CNS
leads to paralysis
it is unusual for poliovirus to escape the gut and enter the CNS This has no benefit for the virus, for the route of exit to find new hosts is via the gut.
What affects the outcome of a viral infection
virus dose, the route of infection, and the age, sex and physiological state of the host.
How does viral dose affect outcome
Low doses are less likely to establish productive infection and cause disease.
High doses, on the other hand, may overwhelm the local innate response and cause disease.
How does route of entry affect the outcome of a viral infection
eg?
The same dose of virus given by different routes can give different
outcomes.
eg the use of variola virus (the poxvirus that causes smallpox) to prevent severe disease if given by dermal infection (variolation), rather than by inhaling the virus naturally (respiratory infection).
Give 3 examples of viruses which have different outcomes depending on the age or sex of the host
varicella zoster virus
hepatitis B
EBV
How is varicella zoster virus affected by age
causes chickenpox after the primary
infection. Infection with VZV in early childhood is generally a mild infection, but it is much more serious as an adult
How does EBV infection differ with age
Usually EBV
infection in childhood is asymptomatic, but if infection is acquired as a young adult the
outcome may be glandular fever (infectious mononucleosis).
How does age and sex affect the outcome from a Hep B infected patient
HBV infection of a
neonate, from an HBV-infected
mother, gives a much greater chance of establishing a chronic infection
than if the infection is acquired later in
life, when the outcome is more likely to be an acute infection
for males it is worse than females. So if a male is born to an HBV-infected mother, becomes infected and no action is taken, he has a 90% chance of developing chronic HBV infection, and a 50% chance of dying from liver cancer due to this chronic HBV infection.
What can a chronic HBV infection lead to
strong chance of developing into liver
cancer (hepatocellular carcinoma,
HCC)
What is the difference in HBV outcome between the sexes
if a male is born to an HBV-infected mother, becomes infected and no action is taken, he has a 90% chance of developing chronic HBV infection, and a 50% chance of dying from liver cancer due to this chronic HBV infection
How does the physiological state of the host affect the outcome of a viral infection
Stress and immunological deficiency are both factors that increase
the likelihood of severe infection.
Both these conditions contribute to the frequency of
reactivation of herpesvirus infections, for instance.
How do we know varicella zoster virus is more potent in stressed hosts
From NASA: there is an increase in varicella zoster virus in the saliva of astronauts during space travel.
Why can FMDV, VZV, measles and rubella not be classified by their target organ
all acquired by the respiratory route, but they would not be described as ‘respiratory infections’.
What happens in superficial infections
Virus replication occurs in the epithelium at the initial infection site (portal of entry) but does not spread to other tissues
they are acute with a short incubation period and short duration period
Give 5 features of a superficial virus infection
remain at portal of entry
acute
short incubation period
short duration
independent of specific immune responses
Give 3 examples of superficial infections
common cold, influenza, gastroenteritis (rotavirus).
How does a viral systemic infection differ from a superficial one
The virus replicates at the portal of entry but then spreads by various routes (lymphatics,
bloodstream, nerves) to other sites where further replication occurs. These infections have a longer incubation period, are more severe, and the outcome is very dependent on specific immune responses (especially CTL).
name 4 viral systemic infections
smallpox, measles, chickenpox, foot and mouth disease
Which virus have we used to define the characteristics of systemic infection
ectromelia virus (the cause of mousepox)
this still remains the paradigm
Describe the route of systemic mousepox infection (6)
enters and replicates in epithelium
- ->draining lymph node
- -> blood stream
- -> amplification in internal organs eg spleen, liver and vascular endothelium
- -> released in higher titres into blood stream
- -> replicates in lungs and skin of hot for transmission to new hosts (portal of exit)
What is primary and secondary viraemia
primary: first time systemic viral infection enters blood stream after infecting the portal of entry and then passing to the draining lymph node
secondary: when the virus is released into the blood in much greater titres after replicating in the liver etc
What are the most common routes of spread for systemic viral infections?
What else can be used>
blood and lymph
viruses like rabies can use nerve tracts
Why is the common cold only ever superficial
rhinoviruses (common cold) grow well at 32 ºC but not at 37 ºC and so replicate
well only in upper respiratory tract epithelium.
Why does budding affect whether a viral infection is superficial or systemic
influenza buds from apical surface of respiratory epithelial cells and so virions are released into the airways (local). This is determined by trafficking of the virus glycoproteins to that cell surface.
In contrast, a virus that budded from a basal layer (into
tissue) might have greater chance of establishing a systemic infection.
Which factors influence whether a viral infection is superficial or systemic (3 examples)
eg temperature it grows best at
location of budding
interaction with phagocytes
How do interactions with phagocytes affect whether a viral infection is superficial or systemic?
several viruses that cause systemic infection (yellow fever virus and ectromelia virus) can grow in macrophages.
what are the 3 categories for viruses based off their ability to persist in the host
acute
persistent/ chronic
persistent/ latent
What happens usually in acute viral infections
What happens if this does not happen? Use an example to illustrate
In these infections the virus is cleared
after an acute infection. This is the
normal outcome in the normal host
Measles virus mutants can persist in the CNS and cause a chronic demyelinating disease (SSPE) with a frequency of about one per million infection
How long can a chronic viral infection last
Name 2 viruses which almost always establish persistence
for years or even life long
HIV and hep c
Is hep B chronic or acute
cleared after the acute phase in about 90% of normal adults, the
remaining 10% become persistently infected.
In contrast, 90% of infected male neonates become infected chronically.
which herpes viruses establish latency and are not cleared
all
Use VZV as an example to demonstrate the stages of a latent infection
The acute disease is chickenpox (varicella) and, during the subsequent latent infection, the virus genome resides in neurones of sensory ganglia for the lifetime of the host. After many decades the virus may re-appear to cause shingles (zoster).
What is the general feature of herpesvirus infections
some cells are permissive for productive infection, while others are non-permissive (the virus enters the cell but gene expression fails) and latent infection is established in these cells.
How may a latent virus recur
Use herpes simplex as an example
a stimulus or
cell differentiation.
Thus a stimulus to the sensory neurone may allow the replication of herpes simplex virus and the seeding of virus into innervated epithelium to cause a recurrence
How can HCMV become recurred
The differentiation of a latently-infected monocyte to a tissue macrophage
results in production of HCMV and seeding of virus into permissive tissue
Give definitions for the following:
a) latency
b) reactivation
c) recurrence
Latency: Virus in a quiescent state
- Reactivation: Virus replication after change to latently infected cell (cell is now permissive)
- Recurrence: Disease after reactivation, seeding of permissive cells and virus multiplication
Where does the virus generally reach the highest titre?
in the organ from which it is shed
often the portal of entry and portal of exit are
the same
Name 4 factors which affect transmission of a virus to a new host
particle stability,
duration of shedding,
concentration of virus shed,
availability of susceptible hosts.
Compare the virion stability of enveloped and non-enveloped viruses
. Enveloped viruses are less stable than non-enveloped ones. The lipid envelope is fragile and can easily be disrupted.
How does the envelope of an enveloped virus affected infectivity
What does this mean for the spread of such viruses
Since the envelope contains the virus attachment protein(s), loss of the envelope results in loss of infectivity.
Enveloped viruses
are therefore usually spread by close contact, e.g. measles virus, influenza virus, and HSV.
What is the incidence of influenza deaths in an average year
there are 10,000 and 15,000
deaths in an average year from
influenza.
What does the FMDV epidemic in southern England demonstrate
small non-enveloped viruses are very stable outside the host and can be transmitted over long distances.
the 1981 FMDV epidemic in southern England
was caused by virus being blown across the English Channel from France.
Which viruses tend to be stable in water
Give examples
Picornaviruses that are spread by the oro-faecal route (poliovirus, hepatitis A virus)
are stable in water.
Describe the shedding of a virus that causes an acute infection
How does this compare to persistent infections
A virus that causes an acute infection is shed for only a short time and therefore high levels of virus are shed to maximise the chance of finding a new host
a virus that can persist in the host can be shed repeatedly over longer time periods and so lower levels are sufficient.
Describe the released virion concentration of acute infections, using rotavirus as an example
Acute viruses shed virions at high concentrations.
rotavirus is shed in watery diarrhoea at concentrations of 109 p.f.u. / mL.
What are the 2 important factors affecting the availability of susceptible hosts
the population size,
and the availability of other host species
Use an example to illustrate the importance of population size in viral transmission
Measles virus is physically unstable and survives for only a short time outside host. It infects
only humans, is antigenically stable and immunity is lifelong. Epidemics (before
vaccination) occurred every few years, mostly in young school children. Most susceptible
individuals are infected during an epidemic and thereafter most of the population is immune
(herd immunity is high).
How is a virus maintained between epidemics
Use measles to exemplify this
maintained by
small numbers of susceptible hosts
In isolated populations of less than 100,000
measles is eliminated. A good example is the
intermittent epidemics in the Faroe Islands.
After an epidemic the virus disappears and a new epidemic can occur only when i) there
are a sufficient number of new susceptible
hosts, and ii) the virus is introduced from an external source.
Why did small pox and measles not really affect humans until 5-10,000 yrs BC
Only when human populations reached
sufficiently high density (5-10,000 yrs BC) could diseases such as measles and smallpox endure in man.
Name 2 viruses that infect multiple hosts
Yellow fever virus and rabies virus
(YFV infects primates and
mosquitoes; rabies infects many mammals)
Why is it hard to eliminate zoonotic diseases
. Infection of humans is incidental and human herd
immunity has no impact on virus survival.
What are the 3 types of vertical viral transmission
congenital infection
perinatal
germline transmission
What is congenital viral infections
Give 3 examples
(AKA transplacental infections).
virus crosses the placenta and infects the foetus in utero. Examples are: rubella, HIV, human cytomegalovirus
What is perinatal viral transmission
Give 3 examples
infection during birth or from breast milk.
Herpes simplex virus (genital herpes
present in birth canal).
Human cytomegalovirus (present in birth canal or in breast milk).
Hepatitis B virus (probably from blood contact at birth).
What is germline viral transmission
The presence of a retrovirus, as the provirus, in the germ line
enables direct transmission to offspring.
virus is transmitted as an inheritable genetic element.
These are called endogenous retroviruses.
Are endogenous retroviruses common
Are they worrying
yes - most species carry them
They are usually silent, but may reactivate under some
circumstances (hence the concerns about endogenous retroviruses and xenografts).
Can herpesviruses achieve vertical transmission
some eg human herpes virus 6 (HHV6) can
integrate an entire virus genome into telomeric regions of chromosomes in cells (including
germ cells) and thereby achieve vertical transmission
True or false
all viruses have a single portal of entry and exit
false: Most of the viruses mentioned above have single entry and exit portals and hence single
transmission routes, but this is not always the case.
Name a virus that has multiple entry and exit portals
what are the portals
human cytomegalovirus can be acquired congenitally, perinatally, by oral contact or by sexual transmission. In addition, the virus can be transmitted by blood transfusion or organ transplantation.
Does the entry portal of HCMV affect its spread
Regardless of the route of entry, the virus infection becomes systemic and grows at multiple exit portals - the salivary gland, the genital tract, in mammary glands and it also crosses the placenta.
How infectious are the viruses transmitted by
usually highly contagious and cause widespread epidemics in which many or most susceptible individuals become infected.
High levels of herd immunity are established and new epidemics must await either: (a) the introduction of sufficient numbers of new susceptible individuals into the population (e.g.,
measles virus) or (b) the appearance of new antigenic variants that can evade herd
immunity (e.g., influenza virus).
Describe the infectivity of viruses secreted in saliva
less contagious and frequently require
contact to achieve transmission (herpes simplex virus; Epstein-Barr virus).
How do food/water infections occur
as sporadic outbreaks associated
with a contaminated source.
In communities where clean water is not available or food hygiene is poor, the incidence of infection and the ‘virus load’ in the community are high.
True or false
Most viruses cause very little disease
true
they have co-evolved with their hosts and reached an evolutionary balance.
Where do human virus pandemics usually come from
What do they often have
give some examples
often zoonotic
high morbidity/mortality
AIDS, severe acquired respiratory syndrome (SARS, caused by a coronavirus),
influenza and Ebola haemorrhagic fever.
What is disease usually caused by
destruction of specific cell types giving rise to the
particular symptoms deriving from loss of those cells, or from immune pathology caused by an
in appropriate immune response to virus infection
describe the impact of the FMDV epidemic (4)
FMDV caused a massive epidemic in UK in 2001 with > 2,000 confirmed cases.
More than 6m
cows and sheep were killed.
Estimated to have cost the UK economy £8b.
A virus just 20 nm in
diameter caused postponement of the 2001 general election
What causes yellow fever
What does it infect
a flavivirus that originated in Africa and was taken to the Americas via European colonisation and the slave trade
primates, mosquitoes and humans and
causes yellow fever (yellow jack)
What causes transmission of yellow fever
Transmission by the female mosquito Aedes aegypti.
What was the impact of yellow fever on colonisation
Greatly
feared by European settlers: very susceptible with high mortality rates.
What vaccine is used for yellow fever
. A live attenuated vaccine (17D) produced
by Max Theiler in 1937: still in use today
Describe the YFV epidemic in Saint Domingue
French colony in the late
18th century. After the French Revolution, slavery was abolished in France and all its colonies
(1795). Napoleon wished to re-introduce slavery in Saint Dominigue, which had been highly
lucrative. Resisted by the emancipated slaves. So in 1802 Napoleon sent ~30,000 soldiers led by
General Charles Leclerc (brother-in-law). Yellow fever killed ~85% of them
What did the YFV outbreak in Napoleon’s armies lead to
Contributed to decision of Napoleon to sell Louisiana to USA in 1803. The Louisiana Purchase
What is myxoma caused by
myxoma virus, a poxvirus whose natural host is the South American rabbit in which it causes relatively little disease
What is the history of myxoma virus in Australia
introduced into Australia to control the European rabbit which, in this
foreign environment, had replicated out of control (500m rabbits = consumption of 1.8b kg of
vegetation / yr). But myxoma virus quickly mutated to become less virulent. Now infection killed
fewer rabbits and more slowly, so the probability of the virus being transmitted increased because
the host survived for longer.
Which viruses does evolution favour
viruses with low/modest virulence
What is the R value of a given virus dependent upon
eg population density and behaviour
Describe the genome of influenza
-ve sense ssRNA
genome with 8 RNA segments
Describe the structure of the influenza virion
Helical nucleocapsid within a lipid membrane
containing the haemagglutinin (HA),
neuraminidase (NA) glycoproteins and M2, an ion channel.
Beneath the virus envelope is the matrix (M1) protein and within the nucleocapsid the nucleoprotein (NP) is associated with the RNA genome.
Virion core also has PB1, PB2 and PA that
make up the virus RNA-dependent RNA
polymerase (RdRp)
Does the RdRp enzyme need to be package with the virion for influenza
The RdRp must be
packaged within the virion to transcribe the –ve sense ssRNA genome into the virus mRNAs to initiate the infectious cycle
What is the shape on an influenza virion
vivo vs vitro
variable but roughly
spherical shape for most influenza viruses grown in cell culture
However, fresh
clinical isolates can have a filamentous morphology
Where does influenza replicate? Is this normal?
Unlike most –ve sense ssRNA viruses, influenza virus replicates in the nucleus
What kind of virus is influenza
Orthomyxovirus
What are the different types of influenza
4 types (A, B, C and D): type A viruses cause most disease in man
How are influenza isolates named?
Hence, what is virus isolate A/PR/8/34
What is another way to name a virus
d according to the type (A, B, C), country of isolation, isolate number, and year.
Thus A/PR/8/34 means the virus was type A, isolated from Puerto Rico, isolate 8 in 1934
according to
their HA and NA proteins, so influenza virus is called H1N1 if it has the H1 HA and N1 HA.
How many HA and NA types of virus are there that are type A influenza
18 HA types and 11 NA types
Where are most HA influenza subtypes found
in birds that are the natural reservoirs of most influenza viruses.
How is influenza transmitted
How does incidence vary
via aerosol and inhaled virions initiate infection in the respiratory tract.
Influenza transmission is highly seasonal with epidemics occurring during winter months when people are closer together inside
Describe the structure of HA
trimer, where each monomer is composed of HA1 and HA2 subunits
HA1 subunit represents the globular head distal to the virus membrane
and includes the sialic acid binding site
HA2 subunit provides the stalk between the globular head and the virus membrane, in which it is anchored.
HA2 also has the fusion peptide at N terminal
What is the HA2 fusion peptide
At the N terminus of HA2 there is a hydrophobic peptide (the fusion peptide) that is needed for fusion.
How does the fusion peptide on HA2 change to promote fusion
At neutral pH fusion peptide is buried within the HA trimer, but upon acidification the HA changes its conformation, the HA1 subunit moves aside, and the HA2 fusion peptide is exposed, enabling insertion into the
endosomal membrane.
This destabilises the membrane and
promotes fusion between the viral envelope and endosomal
membrane
Why is pH important for influenza entry
How is this exploited therapeutically
entry requires low pH to cause fusion, mediated by HA, and to destabilise the core, mediated by M2
Drugs amantadine and rimantidine inhibit influenza virus entry by blocking the M2 proton channel
What are amantadine and rimantidine designed to do
What is the problem with these drugs
e inhibit influenza virus entry by blocking the M2 proton channel.
However, resistance to these drugs is generated rapidly by M2 mutations
What happens to the infleunza virion once it has entered the cell
y the nucleocapsid is transported into the nucleus.
Here the virus RdRp transcribes each –ve sense ssRNA segment into a mRNA
What do the 8 RNA segments do in the influenza genome
In general each segment codes for one protein, but for RNA segments 7 and 8, the mRNA may be spliced to produce different proteins
(M1 and M2 from segment 7, and NS1 and NS2 from segment 8)
Describe the ends of the virus mRNA for influenza
At the 5’ end they have a cap and a few nucleotides derived from cellular
mRNAs.
At the 3’ end the virus mRNAs terminate before the end of the vRNA template and have a poly A tail.
Where is the 5’ cap of influenza mRNA derived from
What does this mean
derived by cap-snatching from host mRNAs
host DNA-dependent RNA
polymerase II activity is needed for influenza replication – to provide the supply of caps that
influenza virus RdRp steals to synthesise virus mRNAs.
What is needed to replicate the influenza genome
how are these produced
What happens once the genome is replicated
complete copies of the genome vRNAs are needed.
These +ve ssRNAs are also produced by the virus RdRp
and are then copied back into more –ve ssRNAs
The
new –ve ssRNAs may be either packaged into new
virions or act as templates from which more mRNA can be made
What happens to the new influenza nucelocapsid after replication and protein production has occurred
leaves the nucleus, moves to the cell periphery and buds through the membrane to acquire its envelope
How do proteins produced by the influenza virus that are required for the new nucleocapsid reach the virus envelope
transported
independently to the plasma membrane.
What is required for the influenza virus to be released from the cell
requires the neuraminidase (NA) that cleaves sialic acid residues from proteins.
What would happen to influenza particles if no NA were present
Since sialic acid is present over the cell surface, if there was no NA, the virus would just
bind back to the cell and be unable to escape
What does the influenza virion do after cleavage of sialic acid by NA
The NA also removes sialic acid residues from the HA and NA proteins on virions, to prevent aggregation of virions.
Which anti-influenza drugs inhibit NA
tamiflu (oseltamivir phosphate) and relenza
What is Tamiflu
What are its effects
analogue of sialic acid and inhibits neuraminidase
influenza virus cannot be released from the infected cell. The virus also clumps to itself because HA and NA are glycoproteins.
What do new influenza drugs target that have recently been licensed
target the cap-dependent endonuclease activity
How does influenza cause repeat infections generally?
The virus undergoes antigenic variation to escape existing neutralising Abs. It can do this in 2
ways: antigenic drift, antigenic shift
animal reservoirs
Name 2 flus from animal reservoirs
H5N1 bird flu, or H1N1 swine flu
What is antigenic drift in influenza
HA gradually accumulates mutations so neutralising Abs cannot bind (selection pressure)
What is antigenic shift
more radical change to the HA caused by acquisition of a
completely new HA from another
influenza virus.
What can happen if a cell is infected with 2 influenza viruses, say a human virus and an avian virus?
|When is this dangerous?
both sets of RNA segments are transported to the nucleus and replicated. During the packaging of RNA segments into new virions, reassortment can
occur so that viruses emerge with RNA segments derived from both parent viruses.
if the progeny virus has derived 7 of the genes from the human virus and the HA gene from the avian virus, the virus can replicate well in man but the existing immunity to HA is of no value
can replicate unchecked, leading to pandemics
What was the most devastating influenza pandemic
1918/9 when 40 - 50 million people died
in 1957 both the HA and HA changed, but in 1968 only the HA subtype changed from the virus circulating previously. What did this show?
antibody to HA is critical in preventing influenza virus infection and antibody to NA is less important
Why was the H1N1 strain that appeared in 2009 more manageable than in 1918
had a low mortality rate (<0.02 %) that compares
favourably with the mortality rate of 2.5% for the 1918/9 H1N1 strain.
What is an influenza virus that currently exists which could become v dangerous
H5N1 - epidemics in birds
highly virulent: human mortality rate of 60%
not shown human-human transmission. If the virus acquired this it would be very dangerous
furthermore, Avian influenza virus NS1 proteins are well adapted to do this in the avian system, but may be less efficient in humans. So the NS1 protein may adapt and become a
more potent inhibitor of human IFN system and so increase virus virulence for man.
What are factors that affect whether an influenza virus can adapt to man
better binding to human cells, better replication in those cells, better escape from human innate immunity better transmission between humans
What is sialic acid linked to
Why is this important
the terminal galactose via either an α2’-3’ or α2’-6’ linkage
HAs from human and avian viruses bind these with different specificity
What sialic acid linkage do human influenza viruses prefer
bird influenza?
α2’-6’ linkage,
whereas avian HAs prefer α2’-3’.
How small does a mutation have to be to cause a change in sialic acid binding
single amino acid changes (e.g. L226Q for the H3 HA)
What influences whether the influenza virus replicates well in humans
Specific amino acids in the PB2 subunit of the RNA polymerase
What happened to avian influenza strains that have had E627 changed to K627 experimentally
replicate well in human cells,
avian viruses that have adapted to man have acquired
this change naturally.
|What confers IFN resistance to influenza
non-structural protein 1 (NS1)
What type of virus is hep A
Describe its genome
how does it express its proteins? what is this similar to?
picornavirus
+ve ssRNA genome
by polyprotein processing, similar to poliovirus
Describe the hepatitis A virion
27-32 nm icosahedron. Non-enveloped
How do you catch hepatitis A
by ingestion of food or water contaminated with faecal material. It is prevented by good hygiene.
How does hepatitis reach the liver
infects epithelial cells of the oropharynx or intestine and spreads to bloodstream (viraemia)
to reach the liver to infect hepatocytes and liver macrophages (Kupffer cells).
How does hep A transmit to another person
Virions are released
into the bile and thereby the faeces
What is a common feature of hepatitis A
jaundice
How does the impact of hepatitis A vary by age
In 90% of children the infection is asymptomatic. In contrast, 80% of adults develop acute hepatitis and may have fever, fatigue,
appetite loss,
diarrhoea and jaundice
Is there a vaccine for hepatitis A
yes, 2:
A live attenuated virus that replicates and induces immunity without inducing disease.
• An inactivated virus preparation
What kind of virus is Hep B
hepadnavirus
What is a hepadnavirus
a small DNA virus that replicates via reverse transcription
eg hepatitis B
What are the different outcomes of hepatitis B
causes acute and chronic hepatitis and hepatocellular carcinoma (HCC
How many people are currently infected with HBV
300 million
How was HBV discovered
Blumberg identified an abundant, unusual antigen in Australian
Aboriginals. the presence of this antigen correlated with chronic hepatitis and was present in
those who developed acute hepatitis naturally or after a blood transfusion. The antigen was named Australia antigen and represents the surface protein of HBV (HBsAg).
What did the discovery of hepatitis B lead to 94)
• the identification of a causative agent of infectious hepatitis, now called HBV,
• the development of a diagnostic method to screen all blood donations for the presence of HBV
and so make a huge reduction to the incidence of post transfusion infectious hepatitis,
• the enactment of legislation making this screening mandatory
• the development of an HBV vaccine, used globally to reduce HBV infection and liver cancer
Describe the HBV virion
e 42 nm diameter, called Dane particles.
They contain an icosahedral capsid surrounded
by a lipid envelope
In a micrograph of HBV virions, what will you be able to see other than the Dane particles
smaller, numerous 22 nm particles. These are lipoprotein complexes containing a lipid membrane and the virus surface protein (HBsAg).
These can also have pleomorphic shape and size and be filamentous. They lack nucleic acid and so are non infectious
How big is the HBV genome
Describe it
3.2 kb
packaged into virions is circular DNA, partially ds, and has 4 overlapping open reading frames, so making efficient use of the coding potential.
What do the following genes in the HBV genome encode
a) core gene
b) polymerase gene
c) HBsAg gene
d) ORF X
a) encodes the capsid protein,
b) polymerase gene encodes the reverse transcriptase (covers the majority of the genome),
c) encodes the surface glycoprotein
(HBsAg) that comes in 3 forms with differing N
termini,
d) ORF X encodes a transactivating
protein.
What is the target for antibodies against HBV
HBsAg binds to hepatocytes to start infection, is
the target for neutralising antibody and is the antigen used in the HBV vaccine
How will a patient’s blood indicate they have HBV
In chronically infected patients this antigen is at very high
concentration in the blood (~ 1% of total serum protein).
Describe the replication of HBV
HBV is a reversivirus meaning that it replicates its genome via a reverse transcription step and it shares this property with the retroviruses
How does HBV replication differ from a retrovirus (2)
i) it packages DNA, not RNA, into the virus particle, and
ii) it does not integrate its genome into the chromosome of the infected cell as part of its
replication cycle, whereas this is an obligate step for retroviruses.
Why is the study of HBV replication so difficult
what else did this fact affect?
When were these problems overcome
cannot be grown in tissue culture
This prevented development of a live attenuated vaccine by passage of the virus in cell culture
by the cloning of the genome in 1978 and its sequencing in 1979. For vaccine development this had great significance.
What are the different forms of HBV transmission
- infected mother to child, at or close to birth,
- infected mother or siblings during the first years of life
- infected blood products
- contaminated needles (drug addicts)
How can a baby be protected from HBV infection transmitted from the infected mother?
vaccination shortly after birth
What is the outcome of acute HBV
h jaundice that resolves and is followed by seroconversion and clearance of virus and of HBsAg from the blood
What is the result of a chronic HBV infection
the virus is not cleared, HBsAg is present at high levels, and there is a persistence liver disease and inflammation that predisposes to HCC.
What influences whether a HBV infection is chronic or acute
age at which infection is acquired, the
sex of infected person and limitations on virus clearance, such as immunological deficiency
How does age of infection with HBV affect disease outcome
90% of those infected at or close to birth develop a chronic infection. Whereas only 10% of those infected when > 5 years old do so
How does sex affect outcome of HBV
Males are more likely to develop chronic infection, and therefore HCC, than females.
What are the current procedures in place to prevent spread of HBV
- Vaccination
* Screening of all blood samples and blood products.
How was the first HBV vaccine made
y purifying 22 nm HBsAg particles from the plasma of chronicallyinfected people. After treatment to remove all infectivity, and conjugation with adjuvant, the
subunit vaccine was injected to induce an antibody response
What were the issues with the first HBV vaccine
- Expensive
- Limited in supply
- Dangerous to make: handling large volumes of infected plasma was risky and there was reasonable concern that other pathogens might also be present in HBV-infected patients
How were the problems with the first HBV vaccine overcome
cloning of the HBV genome
HBsAg was expressed in yeast and purified without concern of infection
What are 3 results of Taiwan’s adoption of universal HBV vacciantion
Reduction in infection of children
• Reduction in chronic infection from >10% to 1% of population
• Reduction in the incidence of HCC
What drugs can be used to treat HBV
Treatment for chronically infected people requires effective drugs or some immune therapy.
There are some dugs but these are not very effective.
How did we know there was another form of hepatitis that wasnt A or B
• post transfusion hepatitis using blood that was negative for HAV and HBV, post transfusion
non-A, non-B hepatitis (PT-NANBH)
• by epidemics of NANBH (E-NANBH
Give 2 experiments that were performed on HCV before they knew what it was
Infection could be transmitted to primates, the agent passed through an 80 nm, but not 30 nm, filter
sensitive to chloroform (i.e. was enveloped).
When the HCV genome was sequenced, what kind of virus was it discovered to be?
flavivirus
Describe the features of the HCV particle
A flavivirus 50 nm diameter Icosahedral capsid (C protein) Enveloped Surface glycoproteins E1 and E2. Genome +ve ssRNA 9.5 kb.
Why have vaccines not been developed against HCV
There is huge diversity in the HCV genome with 6 different clades (up to 30% nucleotide
diversity) and rapid evolution within clades
How does HCV replication occur in vivo
New virions are formed by internal budding into the endoplasmic reticulum and are released by exocytosis.
Can HCV be studied in cell culture?
HCV in cell culture was not possible for a long time,
but virus clone that does replicate has been identified and has been invaluable for studying the replication cycle and for testing of anti-viral drugs.
What are the possible outcomes of HCV
chronic or acute
chronic infection is by far
the more common outcome and to be expected in >70% of cases.
What can happen in chronic HCV infections
After 10-30 years there may be liver cirrhosis in 10-20% of cases, and thereafter liver cancer
in 1-5% of these cases per year.
How many people are currently infected by HCV
170 million
How can we limit HCV spread
screening of blood products and the consequential removal of HCV from blood has eliminated post transfusion hepatitis. This has been a huge success.
What are possible treatments for HCV
originally used IFN (modest success)
new drugs target non structural proteins
these can be used in combination reduce resistance
What are the targets of drugs which attack HCV non structural proteins (4)
NS2 and NS3 proteases,
NS5A
the NS5B RNA polymerase
What is the effect of anti-HCV drugs
Cause a dramatic and
rapid reduction of virus burden and recovery of liver function, indeed they eliminate the
virus. Thus the patient is cured.
What is a key issue with anti-HCV drugs
Currently, a 12-week course of drugs will cost about £60K and so are prohibitively expensive in many countries
Contrast HBV and HCV
genome, variability, control
• HBV
DNA virus
less variable
controlled by an effective vaccine, No effective drugs
• HCV
RNA virus
hugely variable and a vaccine is not available.
drugs can cure infection
What are prions
infectious proteins
What do prions do
induce transmissible spongiform encephalopathies (TSEs) -
chronic, progressive neurodegenerative diseases that afflict both man and animals and are invariably fatal.
Name 4 diseases caused by prions
- Scrapie in sheep.
- Kuru in the Fore tribes in Papua New Guinea, transmitted by cannibalism.
- Creutzfeldt-Jakob disease (CJD) and variant CJD (vCJD) in humans.
- Bovine spongiform encephalopathy (BSE) in cattle (also called mad cow disease)
How did Alders and Gajdusek show kuru was infectious
were able to transmit the disease from man to chimpanzee by injection of post-mortem brain
material from a victim of kuru
How did kuru lead to the prion hypothesis
able to transmit the disease from man to chimpanzee by injection of post-mortem brain material from a victim of kuru
failed to find a virus or bacterium
The infectivity was not destroyed by irradiation that would destroy nucleic acids
What causes kuru
How does it work briefly
PrP
PrP can adopt different
conformations and one of these causes the neurological illness, as it accumulates
What is the conformation of PrP that causes scrapie called
What about the normal conformation
PrP^sc
PrP^c (c is for cellular)
Describe PrP^c
What is its structure
Where is it found
a GPI-anchored protein that is highly conserved in mammals
largely alpha helical, glycosylated and present at the cell surface
most abundant in
the CNS, but also found in the peripheral lymphoid system
Describe the structure of PrP^sc
mostly beta-sheets that is very stable and resistant to protease digestion. This form acts as a template upon which the PrP^C
can refold to form PrP^Sc and so catalyses this conversion
can accumulate and cause disease
What is the conversion of PrP^c to PrP^sc caused/ influenced by? (3)
- It can convert spontaneously (rare)
- The misfolded protein will cause the normal protein to misfold
- Specific amino acids influence the ease of conversion from the normal to the misfolded form.
transmission is influenced by dose eaten
Name 3 prion diseases that are transmitted by ingestion of infected material
Cannibalism – kuru
Eating infected animal food – BSE in cattle
Eating infected animals – nvCJD in man
Are prion diseases always transmitted?
Prion diseases may also be sporadic or familial
What was the BSE epidemic caused by
feeding cattle with meat and bone meal (MBM) infected with
prions, derived from scrapie-infected sheep
Whihc cows were affected by BSE
BSE occured in cows 4-6 years old who had been fed with MBM infected with prions
When was the BSE epidemic
The first cases were detected in Nov 1986 and the number of cases increased rapidly to peak in 1992 with >3000 cases/month. UK had ~ 200,000 cases of BSE and 4.4 m cattle were slaughtered to control the epidemic that was mostly over by 2000.
Can BSE affect humans
It was suggested that BSE couldn’t transmit to man, but it did.
How common are TSEs in man
1 per million people / year.
What is the most common TSE in man
How can it be caused
CDJ
sporadic (90% of cases), familial (10%) or iatrogenic.
How were CDJ cases (appearing 10 years after the BSE epidemic) similar to BSE
- the disease had florid plaques in the cerebellum, like BSE prions in primates
- the PrP glycoform typing showed the same pattern as BSE
By 2014, how many cases of vCDJ were there in UK?
Who was it mostly
How did they catch it
177
young people who had no medical or
surgical risk factors for CJD
probably were infected by eating BSE-infected meat.
Where was CDJ popping up in 2014
177 cases in UK There were 52 cases elsewhere, mostly W Europe, caused by BSE-infected meat from UK.
How can we detect prions (4)
no adaptive immune response to the abnormal form of the PrP, so no immunological
evidence of exposure.
However, there are mAbs that detect PrPSc and distinguish this from PrPC.
The PrP form can also be distinguished by protease digestion followed by immunoblotting of the different glycoforms.
Infectious prions can be detected by bio-assay: namely the induction of disease by prion-infected
material in mice.
What is scrapie
a disease in sheep
Describe the disease progression of scrapie
During the pre-clinical phase the
PrPSc is first associated with gut lymphoid tissue. Later, PrPSc appears in peripheral lymph nodes
suggesting a haematogenic distribution. Finally, PrPSc accumulates in the CNS.
What is the national scrapie plan
In UK there is a successful breeding programme developing flocks that are genetically more
resistant to scrapie
How many coronavirus epidemics have there been
What are they caused by
3
The others were SARS-CoV (2003) and Middle East respiratory syndrome coronavirus
(MERS-CoV) (2012)
CoVs that transmitted from animals (zoonoses). Four other human CoVs have caused mostly mild, common cold-like illnesses
Describe the SARS-CoV-2 virion
enveloped, 80-140 nm diameter, and has prominent surface spikes, each representing a trimer of the spike (S) protein.
Other virion proteins are membrane (M), envelope (E) and nucleocapsid (N), the latter associated with the RNA genome
Describe the genome of CoVs
+ve ssRNA genomes: the largest for animal RNA viruses.
How big is the SARS-CoV-2 genome
What does this genome size require
29.9kb
Like all RNA viruses with genomes of > ~ 20 kb, the CoVs
encode a proof-reading machinery to detect and repair errors introduced during genome replication
What are the proof-reading mechanisms in the SARS-CoV-2
The non-structural protein (nsp) nsp14 (an exonuclease, ExoN) and nsp10,
detect and excise 3’ nucleotide mismatches.
What does the SARS-CoV-2 genome encode
5’ to 3’
most of the genome (~21 kb) encodes the nsps 1a and 1b. These large polyproteins are cleaved into several mature proteins.
The next gene (~3.8 kb) encodes the S protein.
Other structural proteins (E, M and N) are encoded towards the genome 3’ end.
genome also encodes many nsps for genome replication and immune evasion.
Describe the rate of change of SARS-CoV-2 genome
What does this mean
What is one way to promote CoV evolution
due to proof-reading, slower rate of change compared to other RNA viruses
making escape from immunity induced by prior infection or vaccination less likely
Co-infection of the same cell by related CoVs can lead to recombination during replication to form hybrid viruses
What is the most variable CoV gene
one encoding S
What is the CoV spike protein
What does it do (2)
forms homotrimers on the virion surface
mediates i)
binding to target cells via ACE-2, and ii) membrane fusion. It is also the target of neutralising antibodies.
What are the closest relatives to SARS - CoV-2 in the wild
Bat RaTG13-CoV and Bat RmYN02-CoV, both came from bats in Yunnan province, China
How are the following related to SARS-CoV-2:
RaTG13-CoV
RmYN02-CoV
RaTG13-CoV has 96.3% nucleotide identity with SARS-CoV-2 across the whole genome
• RmYN02-CoV has ~97% identity with SARS-CoV-2 in the ORF1ab, although is more divergent in other regions: indicating recombination.
How realted are SARS-CoV-2 and SARS-CoV (3)
share ~79% nucleotide identity
taxonomically are considered
strains of the same virus species.
They bind to the same receptor (ACE-2) due to conservation
of critical aa residues within the RBD of S1
Do bat CoVs bind ACE-2
What does this suggest
the RBDs of S1 of Bat RaTG13-CoV and Bat RmYN02-CoV are quite divergent and so do not bind ACE-2.
not the immediate ancestor
of SARS-CoV-2
Which bats have similar CoVs to COVID-19
Horseshoe bats
Why does SARS-CoV-2 have a greater affinity interaction to human cells than bat CoVs (and SARS-CoV
there is an insertion in SARS-CoV-2 of 4 aa, PRRA, that creates a polybasic furin cleavage site
between the S1 and S2 subunits.
Cleavage here enables increased exposure of the RBD and, thereby, a high affinity interaction with the human ACE-2 receptor
Other than horseshoe bats, which animal has similar CoVs to SARS-CoV-2
How similar are they
Malayan pangolins introduced
into Guangdong and Guangxi provinces, China
shares 91%
nucleotide identity with SARS-CoV-2
Are the bat or pangolin CoVs closer to SARS-CoV-2 genetically?
Although, over the whole genome, this Pangolin-CoV is less closely related to SARS-CoV-2 than RaTG13-CoV or RmYN02-CoV are, the aa sequence of the Pangolin-CoV S1 RBD is the closest match to the SARS-CoV-2 RBD.
In particular, 5 aa residues critical for binding ACE-2 are conserved between Pangolin-CoV and SARS-CoV-2.
Were the bat CoVs immediate ancestors of SARS-CoV-2
The distinctive and extensive nucleotide sequence differences between the bat viruses RaTG13-CoV and RmYN02 indicate that neither were the immediate ancestor
What is the most likely origins of SARS-CoV-2
bat virus that is more closely (>99%
nucleotide sequence identity) related to SARS-CoV-2 than either RaTG13-CoV or RmYN02-CoV,
or from a bat virus that was transmitted to humans from an “intermediate” mammalian host
species, possibly following evolution via recombination with other CoVs
Was SARS-CoV-2 made in a lab?
NO!!!
The divergence between SARS-CoV-2 and other known CoVs is sufficient to refute the assertion that the COVID-19 pandemic arose by the release of a known virus (such as RaTG13-CoV) and makes the unsupported claim that SARS-CoV-2 was created artificially
in a laboratory extraordinarily improbable.
How is SARS-CoV-2 transmitted
What does transmission require
by the respiratory route or by touching surfaces containing virus and then transferring virus to mucosal surfaces.
requires close contact and so occurs more easily in confined spaces especially indoors.
What is SARS-CoV-2 disease characterised by
What is long covid-19
by an excessive pulmonary inflammation that causes loss of lung function
Other more
systemic sequelae are known and the long term consequence of infection on lung function or other parameters are still poorly understood
What is the case fatality rate (CFR)
why is this not completely accurate
3%
estimates were before the extent of asymptomatic infections was understood. CFR has been revised downward about 10-fold so ~ 0.3%
Which factors influence CFR of SARS-CoV-2 (6)
- Age : the disease severity increases steeply over 65 yrs
- Obesity: clinical obesity increases severity
- Hypertension
- Diabetes
- Chronic lung conditions (COPD etc)
- Sex: males are more susceptible that females
What public health measure have been implemented to control covid-19 in the absence of a vaccine
- Social distancing
- Wearing personal protective equipment (PPE) such as face masks
- Hand washing
- Quarantine of those infected and the contacts of those infected, including track and trace
Name 3 drug treatments for covid-19
dexamethasone
remdesivir
mAbs
How does dexamethasone affect covid-19
low doses during severe COVID-19 gives increased survival. This antiinflammatory drug reduces lung inflammation and so may improve lung function.
What is remdesivir
inhibitor of the virus RNA-dependent RNA polymerase (RdRp).
How does remdesivir work
It is
given as a monophosphate pro-drug that is converted into a ribonucleoside triphosphate
analogue to inhibit the virus RdRp.
What is benefit and a danger of giving remdesivir to treat COVID-19
It can hasten recovery
May cause liver toxicity.
How are mAbs used against covid
mAbs directed against the S protein
What are the chances of COVID-19 becoming resistant to the drugs used against it
How will drugs be given ideally
remdesivir might develop because the drug is targeting a virus enzyme
dexamethasone is targeting a cellular process and so the virus cannot change to escape this.
several drugs should be given together to reduce the chances of resistance
How was HIV first spotted
an unusual cluster of opportunistic infections in young
men in Los Angeles: such as, pneumocystis carinii, disseminated HCMV, mucosal candida and
chronic HSV. These infections are normally be controlled by the immune system. Most patients
were homosexuals or intravenous drug users, and all had T-lymphocyte dysfunction
Where did the opportunistic infections spread to from the original clusters of homosexuals/ IV drug uses
spread to haemophiliacs, blood transfusion recipients and sexual partners of at risk group
Where is HIV 2 from
West Africa
less virulent than HIV-1
Des HIV infection induce AIDS quickly?
no can take between 2-15 years to develop
How was HIV spread so widely in humans
Patients may be relatively well for a decade after being infected, but are infectious and can transmit the infection to others.
By 1996, how many cases of AIDS had there been and how many died
> 28 m HIV infections, 5.8m cases of AIDS of whom >75% had died
Is there a cure or vaccine for HIV
No
Drugs can block
HIV replication, but the virus is not eliminated from the body and it will come back if the drugs are stopped, or the virus becomes drug resistant. Without drugs death is the outcome.
Are the numbers of HIV cases increasing?
t for every 10 people who died of AIDS in 2019, 24 more were infected, so the number of HIV- infected people is increasing. Since 2010, there has been a 20% increase.
Is HIV passed on to children
In 2019, 85% of the 1.3 m HIV-infected pregnant women received ART (anti-retrovirus therapy) to prevent
transmission to their children.
What kind of virus is HIV and what family does it belong to?
Which features are common to viruses in this family? (2)
retrovirus
lentivirus family
more complex genomes than the standard
retrovirus, and cause slow infections.
Name 4 lentiviruses other than HIV
- visna virus (sheep),
- equine infectious anaemia virus (EIAV),
- feline immunodeficiency virus (FIV)
- simian immunodeficiency virus (SIV)
Describe the structure of the HIV capsid
cone-shaped, composed of gag p24, and surrounded by an envelope with env
proteins gp120 and gp41 (derived by cleavage of a precursor gp160)
What special feature does the HIV capsid’s gp120 have
gp120 is heavily
glycosylated and this glycan shield restricts access by neutralising antibody
Do you use p24 or env proteins gp120 and gp41 for the target antigen for HIV diagnosis
p24
env is quite variable, due to selective pressure from antibody. In contrast, p24 gag is more conserved
Describe the HIV genome
+ve sense, ssRNA genome with a 5’ cap and 3’ poly(A) tail
diploid genome
contains tRNA used to prime reverse transcription to convert the genome into dsDNA, which can integrate into host genome
Why might we think HIV genome acts like mRNA
does it?
a 5’ cap and 3’ poly(A) tail – just like a mRNA – but it does not function as mRNA
What flanks the HIV provirus
a long terminal repeat (unlike the RNA which has a cap and tail)
True or false
HIV only has gag, pol and env
false
, HIV contains other smaller proteins that
have regulatory or immune evasion functions.
describe the proteins made by HIV (not gag, env, or pol) (3)
tat and rev proteins regulate the switch to expression of the capsid and
envelope proteins later during infection.
Vpu is multi-functional: it down-regulates CD4 (the HIV receptor) and blocks NF-κB signalling to restrict innate immunity.
How did HIV pass from homosexuals and IV drug users to haemophiliacs?
, before HIV was identified and diagnostic kits were developed to screen blood products, blood from HIV-infected donors entered blood banks and many haemophiliacs and blood transfusion recipients were infected.
What is the great majority of HIV transmission now?
either heterosexual or from mother to neonates, and approximately equal numbers of males and females are infected.
What are the 3 major goals in reducing transmission of HIV
education, safe sex and anti-retroviral therapy (ART)
What is HIV tropism and disease outcome dependent upon
the specificity of the HIV attachment protein (gp120) for CD4 that is restricted to helper T cells and macrophages/
dendritic cells
What is needed for HIV to enter a cell
CD4 and co-receptor CCR5 / CXCR4
What are CCR5 and CXCR4
CCR5=macrophage tropic
CXCR4= T cell tropic
hydrophobic transmembrane proteins that function as chemokine receptors
HIV must bind to one of these as well as CD4 to enter the helper T cell or macrophage/ dendritic cell
How does the use of CCR5 and CXCR4 by HIV vary
CCR5 usually used early in infection
CXCR4 is used predominantly later, when the patient develops immunodeficiency
How can you be born immune to HIV
A polymorphism in the CCR5 gene of some Caucasians
causes a 32-bp deletion and so production of a truncated, nonfunctional protein
HOWEVER: Homozygotes should not believe they cannot be infected by HIV:
such homozygotes have been infected.
How common is polymorphism in CCR5 in Caucasians
What about other nationalities?
16% are heterozygous and 1% homozygous
for this mutation
This is not found in Africans or Japanese.
Give stats showing CCR5 mutation affects HIV prevalence
Heterozygotes represent 16% of the Caucasians, but only 10% of HIV-infected Caucasians.
What does HIV gene expression require
transcription of the provirus by host
DNA-dependent RNA polymerase II
Where is the HIV virus promoter
within the unique 3 (U3) region of the left long terminal repeat (LTR)
How are the HIV mRNAs spliced early in infection
how does this change as the infection progresses
heavily spliced so that the small regulatory proteins
predominate, eg tat and rev
as smaller proteins grow in concentration, larger mRNAs that have only a single splice or are un-spliced predominate to be sent to cytoplasm and become structural proteins of new virions
How do new HIV virions leave the cell
Does this kill the host cell
what does this mean? (2)
bud through PM
no
- budding can therefore continue for long periods
- budding cell can be recognized and destroyed by CD8+ CTL
Can a HIV infection be latent
What does this mean
yes
Latently-infected cells
provide a reservoir of HIV genomes that can re-seed infection and are the main reason why
HIV infection is very hard to eliminate
What happens when a HIV infection is latent
provirus is not transcribed, so no virus proteins are
expressed and the cell cannot be detected by CD8+ CTL or antibody
why can drugs that block the HIV replication not fully eliminate the virus
the drugs’ targets are not expressed in latent cells
Describe the levels of CD4+ cells throughout a HIV infection
initial acute drop in CD4 cells but this quickly recovers and patient is asymptomatic
CD4 levels then suddenly decrease to zero after years - this is AIDS
Why is there an initial drop in CD4 cells when the patient has just been infected with HIV
Why does the CD4 count recover after this acute fall
`After infection HIV replicates and induces an HIV-specific CTL response. These CTL recognise and clear HIV-infected CD4+ cells
level of infected cells is controlled by CTL and free virions are removed by Abs
What is HIV doing during the asymptomatic phase of infection
Why do levels of CD4 eventually fall again
continues to replicate and is controlled by the CD8+ CTL.
eventually virus escapes from the immune containment and starts to replicate more extensively, thereby causing destruction of more CD4+ cells
as CD4 levels decrease there is less T cell help to maintain immune response from CTLs and infected cells cannot be cleared
How does AIDS arise from HIV
As the CD4+ cell count falls, there is less T-cell help to produce and maintain a vigorous CD8+ CTL response. Therefore, the infected cells cannot be cleared, the virus burden increases, more CD4+ cells are infected and the immune system decays giving rise to immunodeficiency
What usually kills HIV patients
opportunistic infections
What are different disease progressions of HIV
slow progressors (normal progression) long-term non-progressors rapid progressors.
Describe long term non progressor patients
control HIV for long periods despite being infected. They retain
a strong CD8+ CTL response, high CD4+ cell counts and a low virus burden.
Which haplotypes give patients better HIV prognosis
. Heterozygous carriers of certain HLA haplotypes, such as B27 and B57
Describe HIV rapid progressors
poor prognosis
After the initial burst of virus replication, there
is only a weak CD8+ CTL response and so the infected CD4+ cells are not cleared and
so more virus is produced. Therefore the CD4+ cell numbers decline and the patient
develops immunodeficiency quickly
Which haplotypes are associated with a poor prognosis after HIV infection
Heterozygous carriers of HLA B35 and Cw04, develop AIDS more rapidly.
What are the haplotypes associated with the following outcomes after HIV infection:
long-term non-progressors
rapid progressors.
long term non progressors: . Heterozygous carriers of certain HLA haplotypes, such as B27 and B57
rapid progressors: Heterozygous carriers of HLA B35 and Cw04
How have we tried to develop a HIV vaccine
why does it not work
utilising gp120 to induce neutralising antibodies
HIV undergoes rapid antigenic variation so that even if neutralizing Abs are produced to one strain of virus, others appear and escape.
Also tried vaccine against CD8 CTL
Why is HIV so mutable
How mutable is it
because of its error prone reverse transcriptase
it is estimated that in an HIV-infected patient up to 106 different mutant viruses are produced each
day. The opportunity for immune escape is enormous.
What is the rationale behind the CD8 CTL HIV vaccine
Does it work?
prognosis of infected patients with a strong HIV-specific CD8+ CTL response is reasonably good,
suggesting that CD8+ CTLs are beneficial.
cannot prevent infection by free virus particles and antigenic variation can alter the virus peptides that are presented on class I MHC molecules and thereby enable escape from CD8+ CTL-mediated control
Which immune response would have the best chance of defeating HIV
A combination of Ab- and CTL-based immune responses has the best chance of succesS
How many drugs are there for HIV
Name 4 different types
> 25
Fusion inhibitors
• Chain terminators
• Integrase inhibitors
• Protease inhibitors
What is retrovir also called
What does it treat
Azidothymidine (AZT) or zidovudine
HIV
How does azidothymidine work
thymidine
analogue that is used against HIV. AZT is phosphorylated to the NTP by cellular kinases and is incorporated into the virus DNA by reverse transcriptase.
Incorporation terminates chain growth (there is no 3’ OH).
What does the specificity of AZT for HIV infected cells derive from
the fact that it is a better substrate for the HIV reverse transcriptase than it is for the DNA pol of cells.
Name 3 chain terminating analogues used to treat HIV
Dideoxycytidine and dideoxyinosine, 3TC (lamivudine) are other chain terminating nucleoside analogues.
Name 2 HIV protease inhibitors
saquinavir,
ritonavir.
How do HIV protease inhibitors work
The gag and gag-pol proteins are translated as a polyprotein that is cleaved by a virus protease
during virion maturation to yield the capsid proteins and pol.
These drugs prevent the
completion of virus assembly
HIV is highly mutable, so if a single drug is given, the virus changes rapidly and becomes drug
resistant. How does one overcome this?
give multiple drugs simultaneously making it much more difficult for the virus to
mutate to acquire resistance to all the drugs at once. Highly active anti-retroviral therapy
(HAART) has had a dramatic impact on life expectancy and HIV-associated deaths.
How costly is HAART
initally was v expensive because multiple drugs must be used continually for life and so its use was limited by cost
but price has now been reduced
What is the ultimate solution to HIV
prevention rather than treatment or cure.
Currently, there is no vaccine for prevention and no cure once infected.
Give 5 methods of epidemiological control
quarantine/isolation/slaughter surveillance hygiene regulations vector control screening of blood and blood products
Name 2 viruses that were controlled by quarantine/ isolation/ slaughter
Give some detail about each
small pox - eradication utilised vaccination, surveillance and quarantine
rinderpest: was, and foot and mouth disease is, controlled by surveillance and then imposing quarantine on infected areas and slaughter of infected herds
what does surveillance of notifiable diseases enable
Which diseases are controlled in this way
Surveillance enables implementation of public health measures, vaccination or anti-viral drugs
influenza, measles, rubella and
AIDS.
Which viruses can be controlled by hygiene regulations
Viruses spread by the faeco-oral route
are controllable by good hygiene standards and clean water. Eg poliovirus and HAV.
What is vector control
. Those viruses that are spread by mosquitoes such as yellow fever virus, Zika virus and dengue virus, can be controlled by reduction of urban mosquitoes or
protection from them
What is blood screening useful for and what is a drawback?
Which viruses is it useful for?
Rigorous screening of blood and blood products
effectively prevents spread in this way.
But screening is only possible once a pathogen is
known to exist.
Examples are: hepatitis B virus, hepatitis C virus and HIV.
What is the key challenge with developing antiviral chemotherapy?
How does this contrast with antibiotic development
specificity because the virus uses host machinery for many replicative functions
antibiotics target the bacterial ribosome or cell wall biosynthesis, so don’t
affect host.
How can we make antiviral drugs specific to virally infected cells not healthy cells
virus-specific enzymes provide targets: These include nucleic acid polymerases, proteases, neuraminidase (influenza) and HIV integrase.
Give antiviral drugs that are used against the following viruses: influenza HCV HIV HSV
influenza: amantadine against M2, tamiflu against NA
• HCV: several that target NS5A
• HIV: fusion, pol, integrase and protease inhibitors
• HSV: acyclovir, a nucleoside analogue and chain terminator
What are inhibitors of virus polymerases generally
Nucleoside analogues
How does acyclovir work
What is it also called
phosphorylated by HSV thymidine kinase, but not cellular kinases. The NTP is then incorporated into viral DNA by the HSV DNA polymerase,
leading to chain termination. Hence ACV only works in infected cells where virus TK and DNA pol are present.
Zovirax
When did small pox and measles become dangerous to humans
Smallpox and other highly contagious infectious diseases that induced long-lasting immunity, such as measles, only became endemic in man when humans changed from hunter gatherers to farmers and so human population densities increased to provide a constant supply of susceptible hosts for these pathogens.
Which virus causes small pox
variola virus
small pox was the DISEASE
What is a feature of small pox that allows you to distinguish it from chicken pox
in small pox, skin pustules are more abundant on the face than trunk
(“centrifugal” distribution)
What are the 2 types of variola virus
Variola major virus (mortality rate of 30 - 40% in unvaccinated
populations)
variola minor virus (or alastrim) - mortality rate of 1%
What was the first means of controlling smallpox in Europe
How was this introduced?
variolation (or inoculation)
by Mary Wortley-Montague in 1717 who saw the Turks using this in Constantinople.
How did variolation work in 1717?
Pustular material containing infectious virus from a patient who survived smallpox was
inoculated into the skin (arm). This had a better outcome (1% mortality) than acquiring the
infection naturally by respiratory infection (30-40% mortality)
virus could still be transmitted
Why was arm to arm transfer of disease (to allow virus vaccines to travel long distances) banned?
other pathogens could be transmitted simultaneously.
How much did the eradication of small pox cost
Does any small pox still exist
$250 million
in 1996 WHA adopted a resolution to destroy the remaining virus stocks in 1999, variola virus remains in two high security laboratories (USA & Russia) under WHO oversight
Give 6 reasons why we were able to eradicate smallpox
no animal reservoir infection was acute easily recognisable no antigenic variation good vaccine WHO's sheer determination
Why will yellow fever and rabies not be eradicated
animal reservoir - smallpox was only a human disease
Why is the fact that smallpox infection was acute important for its eradication
the virus did not establish latent or persistent infection: contrast
with the herpes viruses
Smallpox was an easily recognised disease: contrast with another disease
HIV
Why was the smallpox vaccine so good
worked against all variants potent as a single dose, low cost and abundant (self-replicating), heat stable when freeze-dried, easy to administer, induced cellular and humoral immunity
Did we know at the time that the smallpox vaccine would work against all strains
No.
The virus genome sequenced only after eradication and then the capsid and envelope proteins of vaccinia virus and variola virus were shown to be highly conserved.
What is the lesson from the smallpox vaccine
To have an effective vaccine and eradicate a disease, you don’t need to understand how it works, rather “if it works, use it”
Can measles be eradicated
Vaccines for measles, mumps, and rubella (combined in MMR) available since 1960s could
eradicate these diseases. But the MMR vaccine is under-utilised due to vaccine hesitancy, in
part due to erroneous claim that it causes autism.
Where was rinderpest important
was of great veterinary importance in Africa where it caused devastating
epidemics in domestic cattle, buffalo and related ungulates.
Give 2 viruses that have been eradicated and 8 that have been controlled all thanks to vaccines
Eradicated: smallpox and rinderpest
Controlled: e.g. diphtheria, tetanus, pertussis, yellow fever, polio, measles, mumps, rubella
Give 8 milestones in vaccine development
1796-smallpox vaccine 1885- Pasteur's development of anthrax and rabies 1937- Theiler developed YFV vaccine 1943- Influenza vaccine 1950s -Polio vaccine 1960s-MMR vaccine 1986 - HBV vaccine 2006 - Genetically engineered vaccine for HPV
Give 10 milestones in the eradication of smallpox
- 1796 Vaccination.
- 1801 Eradication predicted
- 19th century, vaccine spread by arm to arm transfer.
- 1939 UK became free of smallpox.
- 1955 Freeze dried vaccine developed.
- World Health Assembly adopted proposal to eradicate smallpox
- 1967 Intensified eradication campaign. Ring vaccination.
- 1977 Last naturally occurring case (Somalia)
- 1980 Eradication certified by WHA
- 24.2.2015. A collection of variola viruses was destroyed at CDC, USA
What are the 3 different types of vaccine
live, dead (killed) and passive
What re the different types of live vaccine
Give an example of a virus that is controlled by each type
attenuated mutant of virus (eg yellow fever)
live, related virus (vaccinia virus for smallpox)
name 2 live related virus vaccines
vaccinia virus for smallpox,
turkey herpes virus for Marek’s disease (tumour inducing virus of chickens).
What are the advantages and disadvantages of live vaccines
Advantages: self-replicating (so cheaper), induce both cellular and humoral immunity that is long lived.
• Disadvantages: the virus might revert to virulence and might cause problems in
immunocompromised vaccinees. Cold storage is needed for most live vaccines.
Compare the pros and cons of the Salk and Sabin vaccines
both polio vaccines
Sabin (live):
Advantages: self-replicating (so cheaper), induce both cellular and humoral immunity
that is long lived.
• Disadvantages: the virus might revert to virulence and might cause problems in
immunocompromised vaccinees. Cold storage is needed for most live vaccines.
Salk (killed):
Advantage: safety (no infectivity).
• Disadvantages: require multiple administration with adjuvant to achieve adequate level of immunity: mostly induce antibody rather than cellular immunity
What is a killed vaccine
Whole virus that has been inactivated (e.g. poliovirus, Salk)
What are subunit vaccines
contains a component of the virus derived from whole virus (e.g. influenza) or expressed by genetic engineering (e.g. HBV, HPV).
What are the advantages and disadvantages of killed vaccines
- Advantage: safety (no infectivity).
- Disadvantages: require multiple administration with adjuvant to achieve adequate level of immunity: mostly induce antibody rather than cellular immunity.
What is passive immunization
Give 2 examples
Giving preformed antibodies against the pathogen. E.g. serum from immunised animals, now replaced by mAbs of target species
. Examples; after exposure to rabies or HBV (as neonate).
Maternal Abs from breast milk
What are the advantages and disadvantages of passive immunization
- Advantages. Immediate protection (post exposure).
* Disadvantages: serum sickness (formerly), and short lived nature of the protection.
What are 3 things you need to decide when you’re making a vaccine
which antigens
which type of immunity
when is immunity needed
Which antigens are usually targeted for vaccines
Usually a surface protein for neutralising antibody.
What are the different types of immunity a vaccine can provide
Antibody or cell mediated responses. If antibody, should
this be IgA (on mucosal surfaces) or IgG (systemic)?
What are the different times a vaccine needed
When does the pathogen induce disease. E.g. rubella virus is a problem during pregnancy. Vaccinate before travelling to endemic areas. Don’t immunise against measles too soon (maternal Abs may neutralise the live vaccine)
Give 4 approaches for vaccine development
rational attenuation
live recombinant virus
virus like particles
nucleic acid immunization
What is rational attenuation in terms of vaccine development?
Could this apply to SARS-CoV-2?
modification or deletion of a virus gene promoting virulence.
The vaccine for pseudorabies virus (a herpesvirus of pigs) is an engineered vaccine in which the thymidine kinase gene is deleted.
Deletion of ExoN from SARS-CoV-2?
What is live recombinant virus vaccine development
express the gene encoding the desired antigen in a live (safe)
virus vector
eg rabies glycoprotein gene in vaccinia virus
Multiple genes from different pathogens can be engineered into the same virus to create polyvalent vaccines
How does the rabies glycoprotein being transferred to vaccinia virus work as a vaccine
Infection with the
recombinant virus induces immunity to rabies (and smallpox). Used to immunise the foxes
against rabies in parts of Western Europe.
What types of vaccine is the HPV vaccine? How does this work
Virus-like particle vaccine
Synthesis of the capsid protein of some viruses can result in the
production of ‘virus-like particles’
How does nuclei acid immunization work
(AKA Prime-boost)
Inject DNA encoding the antigen under a strong promoter. Boost with a live virus vector expressing the same antigen
What are viruses useful for?
For studying cell biology and immunology
• For gene therapy
• For vaccine development
• For cancer therapy (oncolytic viruses)
Why are retroviruses and hepadnaviruses unusual
Replication involves reverse transcription
Retroviruses synthesis progeny viral RNA via DNA intermediate (RNA->DNA->RNA)
Hepadanoviruses synthesise progeny DNA via RNA intermediate (DNA->RNA-> DNA)
