35. Virology Flashcards
Summarise the discovery of viruses.
What visual techniques are used to study viruses?
[EXTRA]
- Electron microscopy has in the last 50 years developed sufficiently good resolution to view viruses
- X-ray crystallography is also useful in understanding the structure of viruses
What is the size of viruses?
[IMPORTANT]
20nm to 300nm.
Can viruses be detected by a light microscope?
[IMPORTANT]
No
How can bacteria and viruses in a sample be easily differentiated using a physical method?
Viruses can pass through bacterial filters (based on their size).
What is the system for classifying bacteria called and what is it based on?
[IMPORTANT]
- Baltimore system
- Based on the way in which a virus generates mRNA from its genome
Describe the different groups of viruses in the Baltimore classification.
[IMPORTANT]
- Group I -> Similar to humans. Have double-stranded DNA and use RNA polymerase to produce mRNA.
- Group II -> Have single-stranded DNA. Needs to be converted into double-stranded DNA before mRNA can be transcribed.
- Group III -> Have double-stranded RNA and use RNA polymerase to produce mRNA.
- Group IV -> Have single-stranded positive RNA. It must be copied into the negative single-stranded form before it can be transcribed correctly into mRNA.
- Group V -> Have single-stranded negative RNA and use RNA polymerase to produce mRNA.
- Group VI -> Have single-stranded positive RNA. Use reverse transcriptase to produce double-stranded DNA from it. This is then transcribed into mRNA.
- Group VII -> Have double-stranded DNA. This is transcribed by the host cell’s RNA polymerase. In replication, reverse transcriptase is used to renew the DNA genome.
Summarise the genetic material in each group of viruses in the Baltimore classification.
- Group I -> Double-stranded DNA
- Group II -> Single-stranded DNA
- Group III -> Double-stranded RNA
- Group IV -> Single-stranded positive RNA.
- Group V -> Single-stranded negative RNA.
- Group VI -> Single-stranded positive RNA (with reverse transcriptase)
- Group VII -> Double-stranded DNA (elaborate process)
Describe the different levels of classification of viruses that allow us to group them into families.
The features considered in order are:
- Nucleic acid (DNA or RNA)
- Symmetry of capsid
- Presence of envelope
- Number of segments of nucleic acid
- Baltimore class
For each of the Baltimore groups of viruses, name some medically-relevant viruses and the symptoms they cause.
What is a virion?
The infectious form of a virus.
(i.e. It is essentially another name for a virus - CHECK THIS)
Describe the basic structure of a virion.
[IMPORTANT]
- Nucleic acid genome
- Capsid (Protein coat)
- Lipid membrane (some viruses)
What is the capsid of a virion?
The protein that packages the virion’s nucleic acid.
What is the envelope in (some) viruses?
- A lipid bilayer that surrounds the capsid.
- It is derived from the host cell.
- It contains viral attachment proteins.
Explain the concept of positive and negative nucleic acids in viruses.
[IMPORTANT]
- Viruses may contain positive or negative (or both) DNA/RNA
- The polarity refers to the direction of the strand
- The negative strand is the one used as a template for mRNA synthesis
- The positive strand is the one that corresponds in sequence to the mRNA sequence that is transcribed from the negative strand
Explain the concept of segmentation in viruses.
[IMPORTANT]
In viruses, the DNA may be divided into more than one section, which is referred to as it being segmented.
What can destroy a virus’ envelope?
[IMPORTANT]
Detergents, such as bile.
What is the name for how virions replicate?
They are cryptobiotic, which means they do not replicate extracellularly. They are simply carriers of the infection from cell to cell.
What is the eclipse phase?
[IMPORTANT]
It is the time period when a virus has entered a cell, but there has not been a big release of viruses from the cell, so the virus is undetectable.
What is a virus tropism?
A permissive cell or tissue that a virus infects.
What makes a cell permissive for infection (i.e. a virus tropism)?
It must have:
- Viral receptors
- A lack of restriction factors that would inhibit virus growth inside the cell
What is multiplicity of infection (MOI)?
The number of infectious particles per infected cell.
What is burst size?
The number of new infectious particles produced by a bursting virus-infected cell.
What is a defective virus?
A non-infectious virus.
Describe the replication cycle for a virus (such as HIV).
- Attachment to host cell
- Binding to cell-surface receptors
- Release of capsid into the cell
- Uncoating of the capsid, releasing the genome
- Replication of the viral genome
- Transcription of genome
- Translation of genome
- Proteins and other components are assembled into a new virus particle
- New virus is released from the host cell
Can viruses only bind to cell-surface proteins on host cells?
No, they can also bind to carbohydrates, etc.
For HIV-1, state the:
- Virus attachment proteins
- Cellular receptors
[IMPORTANT]
- Virus attachment proteins -> GP120
- Cellular receptors -> CD4 (and co-receptors CXCR4, CCR5)
For SARS-CoV-2, state the:
- Cellular receptors it binds to
[IMPORTANT]
ACE2
What are some co-receptors on host cells that HIV-1 binds to?
[IMPORTANT]
Chemokine receptors CXCR4 and CCR5.
Explain the relationship between the distribution of cellular receptors for a virus and its tissue-tropism.
[IMPORTANT]
The cellular receptors on host cells determine which cells the virus can bind to using its attachment proteins.
By what process are viruses taken into host cells?
Endocytosis, which is usually dependent on receptor binding.
What are the two main different types of infection?
- Acute -> This is when you get ill for a short period, then you recovery from the virus
- Persistent -> This is when the virus is continually present in the body
Describe the properties of a virus causing an acute and persistent infection.
Acute:
- Must have at least some ability to avoid the innate and adaptive immune responses
Persistent:
- Must have immune evasion abilities, PLUS…
- Ability to avoid apoptosis
- Finding immune privileged sites
- Latency by genome persistence
Give some examples of acute and persistent viral infections.
For HSV-1, state the:
- Family
- Genome
- Tropism
- Symptoms
- Pathogenesis
For coronavirus, state the:
- Family
- Genome
- Tropism
- Symptoms
- Mortality
- Vectors
For Marbug/ebola, state the:
- Family
- Genome
- Tropism
- Symptoms
- Mortality
- Cause of death
- Vector
Describe how vaccines for hepatitis B were developed.
- In 1981, the inactivated antigen on hepatitis B cells was purified, allowing it to be used as a vacine
- In 1986, recombinant technology allowed the same antigen to be produced in yeast, for use in a vaccine
Describe how vaccines for HPV were developed.
- The HPV virus consists of a naked capsid
- The vaccine is made by expressing just the capsid, without all of the virulent material inside, so that an immune response can be mounted without endangering the patient
- This protects mucosa from the virus by covering them in IgG
Give an example of an antiviral drug.
[EXTRA?]
Acyclovir:
- Acyclovir has a similar structure to guanosine
- The herpes virus’ thymidine kinase enzyme adds a phosphate to the acyclovir
- The host cell adds more phosphates to give the triphosphate version of the acyclovir
- During DNA synthesis mediated by the infection, the acyclovir triphosphate is incorporated into the chain
- This terminates synthesis because there is no 3’ carbon on the acyclovir, so no more nucleotides can be added to the chain
- This prevents the virus from completing the cycle
This is selectively toxic to the virus because the human cell can differentiate between the guanosine and the acyclovir
How can viruses be made clinically useful?
They can be used as vectors in vaccines.
What is influenza?
- Influenza viruses in humans cause influenza (the flu), an acute self-limiting febrile respiratory infection
- This can involve mild to severe illness, and at times can lead to death
What family do influenza viruses belong to?
Orthomyxoviridae
What are the different types of influenza vaccine?
- Influenza A viruses (humans, birds, pigs, horses, bats, etc.)
- Influenza B virus (humans)
- Influenza C virus (humans)
- Influenza D virus (cattle, goats, pigs)
A and B are responsible for most cases in humans.
Compare the meaning of epidemic and pandemic.
- Epidemic = A disease that affects a large number of people within a community, population, or region.
- Pandemic = An epidemic that’s spread over multiple countries or continents.
Describe the sorts of outbreaks that influenza causes.
Annual seasonal epidemics (caused by influenza A and B viruses):
- Limited outbreaks every winter
- Infection unpleasant but generally non-fatal
- Up to ~ 1 billion infections, with up to 500,000 deaths
Pandemics (caused by influenza A viruses):
- Unpredictable outbreaks that spread rapidly and globally
- Caused by novel influenza viruses emerging from the animal reservoir
- Because of lack of pre-existing immunity in humans more infections and more deaths than usual
- Typically more severe symptoms than in seasonal epidemics
What are the symptoms of seasonal influenza?
What is the site of infection of influenza?
- First infects the upper airway
- Then spreads to the ciliated cells in the bronchus and bronchioli
But it remains restricted to the lungs.
Describe the phases of an influenza infection.
- Short incubation period
- Fever appears around day 2
- High titres of the virus are seen then, meaning the individual is most infectious then
- Innate immune responses typically supress the virus within a week
- Adaptive immune response develops later, preventing subsequent infections
What are some complications that can occur with influenza?
Severe complications include pneumonia. This can be primary viral pneumonia or secondary bacterial pneumonia.
What group is influenza in the Baltimore classification? Describe its genetic material.
- Group V
- This means it has negative RNA, which can be transcribed directly into mRNA by RNA polymerase (which is then used to make the protein)
Describe the morphology of the influenza virus.
In culture:
- Spherical virions
- Around 120nm in diameter
Clinical isolates:
- Filamentous virions
- Around 80nm wide, Over 250nm long
Describe the structure of an influenza A virus.
- Lipid bilayer derived from host cell, containing:
- Haemagglutinin + Neuraminidase -> 2 glycoproteins that are the main antigens
- M2 ion channel (or BM2 protein in influenza B)
- M1 matrix protein layer -> Just below the lipid bilayer, Involved in structure of the virus
- Non-structural proteins 1 + 2 (NS1 and NS2)
- 8 viral ribonucleoprotein complexes -> Contain the negative single-strand RNA of the virus
What is the main influenza types responsible for pandemics? Compare influenza A and B.
- Influenza A is responsible for pandemics, since it has many different subtypes of HA and NA.
- Influenza B only has one type of HA and NA, plus there is an BM2 membrane channel protein instead of the M2 ion channel.
What is the structure of the viral ribonucleoprotein (vRNP) in influenza?
- vRNP contains negative single-strand RNA
- Each of the 8 vRNA segments contains one or two open reading frames
- Each ORF is flanked by a non-coding region that is highly conserved
- The two non-coding regions come together to make a loop of RNA -> The RNA polymerase can interact with this region
- The whole of the RNA molecule is bound to a long nucleoprotein molecule -> The whole structure takes on a double-helix structure
Describe the parts of the RNA polymerase in influenza.
It is a heterotrimer of:
- Polymerase basic 1
- Polymerae basic 2
- Polymerase acidic
What does each of the 8 main vRNA segements in influenza A encode?
[EXTRA?]
How are influenza viruses grouped into subtypes?
Based on their haemagglutinin and neuraminidase surface glycoproteins.
Describe the different types of influenza A and B viruses.
Influenza A:
- There are 18 haemagglutinin (H1-H18) and 11 neuraminidase (N1-N11) subtypes recognised within the influenza type A viruses.
Influenza B:
- There are two lineages, the Victoria and Yamagata.
Describe how influenza isolates are named.
Can seasonal influenza reinfect people?
Yes, because there is a large degree on mutation over time, even within a subtype (such as the H3N2 subtype).
What are the two types of antigenic changes in influenza?
- Antigenic drift
- Small changes in the antigenic structure due to random mutations within the antigen gene, caused for example by the error-prone RNA polymerase
- Lead to epidemics (i.e. yearly seasonal outbreaks of the flu)
- Antigenic shift
- Large changes in the antigens due to obtaining a new gene for the antigens (haemagglutinin and neuraminidase) from e.g. swine flu
- Lead to pandemics
How can antigenic shift happen in influenza?
Reassortment of a virus:
- When two influenza viruses invade a cell at once, they each release their 8 vRNA segments into the cell
- This means that there are theoretically 256 ways for the segements to be rearranged into new viruses
- This is how RNA is transferred from one virus to the other, causing antigenic shift
Describe the generation of new pandemic influenza viruses over time.
It occurs by antigenic shift driven by reassortment of vRNA segments.
What are the two main strands of influenza currently circulating?
- H3N2 -> Hong Kong flu
- H1N1 -> Swine flu
Draw the genesis of the 2009 swine flu (H1N1).
Can avian influenza viruses infect humans?
- Yes, certain avian influenza viruses, such as H5N1 can infect humans.
- However, these tend to be sporadic outbreaks amongst bird-handlers and there is usually no transmission between humans.
- But when there is transmission, it is very dangerous.
Give an example of an avian influenza virus that is very dangerous to humans. Explain why.
- H5N1 avian influenza causes severe multisystem disease in humans
- This is suspected to be partly due to the virus causing excessive activation of the innate immune response
- This leads to inflammation, which leads to problems such as acute respiratory distress syndrome
Explain how an avian influenza virus (e.g. H5N1) could turn into a human pandemic.
- Firstly, the virus infects a human, and although it is dangerous, it is not yet transmissible between humans
- Therefore, it can either:
- Adapt via mutation to become transmissible
- Undergo reassortment (leading to antigenic shift) due to a simultaneous infection with a human virus
- This can lead to a pandemic because there are new antigens that the human population has no immunity against
Diagram is from: (Russell, 2005)
Summarise the 5 main stages of influenza’s life cycle.
- Entry into the cell -> Virus binds to the cell surface proteins via viral attachment proteins, then enters by endocytosis
- Entry of vRNPs into the nucleus
- Transcription and replication of the viral genome
- Export of the vRNPs from the nucleus
- Assembly and budding of new virus at the host cell plasma membrane
What is unusual about the way influenza virus replicates its genetic material?
- It is a negative single-strand RNA virus (Baltimore group 5)
- However, it uses the cell nucleus for transcribing and replicating its RNA
- This is unusual for an RNA virus, which usually have an entirely cytosolic life cycle
What are some advantages of influenza having part of its life cycle in the host nucleus?
It has direct access to nuclear factors, such as splicing factors, for processing its mRNA.
Describe the virus attachment protein and host cell surface receptor involved in binding and entry of influenza.
[IMPORTANT]
- Virus attachment protein -> Haemagglutinin (HA)
- Host cell surface receptor -> Sialic acid (on glycoproteins and glycolipids)
What prevents most avian strains of influenza from infecting humans?
[EXTRA?]
- Human influenza viruses prefer binding to sialic acid attached to galactose via a α-2,6 linkage, while avian influenza viruses prefer binding to sialic acid attached to galactose via a α-2,3 linkage.
- The pig is a “mixing vessel” for both types of virus, since it can be infected by both types.
- Some transmission to humans can also occur because we have sialic acid α-2,3 linkages in the lower respiratory tract.
Where in the body do avian and human influenza strains affect?
- Avian -> Lower respiratory tract -> Because we have sialic acid α-2,3 linkages to galactose in the lower respiratory tract.
- Human -> Upper respiratory tract -> Because we have sialic acid α-2,6 linkages to galactose in the upper respiratory tract.
This partly explains why avian influenza viruses do not spread as easily between humans.
Influenza is taken up into host cells and is then is an endosome. How is its genome released from the endosome?
[IMPORTANT]
- There is gradual acidification of the endosome due to entry of H+
- Two things must now happen
First, the viral envelope and endosome membrane must fuse:
- Acidification causes major structural rearrangement of the haemagglutinin
- This exposes the fusion peptide, a part of the haemagglutinin that enables fusion between the virus envelope and the endosomal membrane
- This allows a pathway to be created, ready for the release of the vRNP complex
Secondly, the vRNP complex must be released:
- H+ ions from the endosome enter the virion via the M2 ion channels (Note: Influenza B has an BM2 membrane channel protein instead)
- This acidification of the virion leads to release of the vRNP complex from the virion
How can the release of influenza’s genome from the host endosome be clinically exploited?
[EXTRA]
- The release of the vRNP complexes from the virion is caused by H+ entry into the virion via M2 ion channels.
- This can be inhibited by M2 blockers, such as amantadine
- However, these are rarely used clinically, since resistance to them has developed in most influenza viruses
(Note: Influenza B has an BM2 protein instead)
What happens after influenza vRNP enters the host nucleus?
It is either:
- Replicated -> This involves the production of an intermediate structure, known as the cRNP (complementary RNP), which then acts as a template to produce more vRNP molecules
- Transcribed -> To produce mRNA that can be translated to produce viral proteins
What enzyme is responsible for the replication and transcription of influenza vRNA in the host nucleus? Draw the structure.
RNA-dependent RNA polymerase (a viral protein)
What is a crucial role of influenza RNA-dependent RNA polymerase?
It has cap-snatching activity:
- The enzyme can remove the first 10 to 20 residues of a host cell’s RNA
- It can then attach this cap to viral RNA to allow it to be translated
Describe the fidelity of influenza RNA-dependent RNA polymerase.
It has a high error rate because it lacks a proofreading mechanism -> This contributes to antigenic drift in seasonal influenza viruses.
Describe how the influenza RNA-dependent RNA can be targeted by antiviral drugs.
The structure has been determined by X-ray crystallography and cryo-EM. This has enabled targeting by various drugs.
Describe what happens after transciption of mRNA from influenza vRNA.
- The mRNA is exported from the nucleus
- In the cytoplasm, it is translated into proteins
- Some of these (such as haemagglutinin and neuraminidase) are go to the plasma membrane
- Some of these (such as the polymerase and nucleoprotein) return to the nucleus, where they are assembled into new vRNP complexes
- These complexes are then also exported to the plasma membrane where they assemble
- The virus is released from the cell by budding
Describe how release of a newly-made influenza virus from the host cell occurs.
It is dependent on neuraminidase, which cleaves sialic acid and therefore causes release of the virus.
How can the release of new influenza viruses from host cells be targeted by antiviral drugs?
[IMPORTANT]
Neuramidinidase inhibitors are used as antiviral drugs, since neuraminidase is required to cleave sialic acid, releasing new viruses.
These are [IMPORTANT]:
- zanamifir (Relenza)
- oseltamivir (Tamiflu)
State the two main types of antineuraminidase drugs (used to treat influenza).
[IMPORTANT]
- zanamifir (Relenza)
- oseltamivir (Tamiflu)
What is the main antiviral mechanism of host cells against influenza?
- The cells can recognise the virus and enter an anti-viral state, which stops or slows down viral replication.
- RNA helicase RIG-I is a sensor for influenza virus RNA, and becomes activated upon binding to influenza virus RNA
- This leads to the expression of interferons and antiviral cytokines
- However, influenza has NS1 (non-stuctural protein 1), which antagonises the interferons:
- By sequestering the viral RNA (hiding it)
- By preventing RIG-I activation
- By interfering with the polyadenylation machinery of the host cell, so that antiviral proteins cannot be produced
Summarise the function of NS1 in influenza.
Non-structural protein 1 is a very potent inhibitor of immunity and allows influenza virus to efficiently escape the immune surveillance and to establish infection in the host.
What are the main types of vaccine against influenza?
- Inactivated influenza vaccine
- Live attenuated influenza vaccine
Describe inactivated influenza vaccines.
- Virus grown in embryonated chicken eggs or cell culture
- After this, it is chemically inactivated
- The main antigen components of the vaccine are the haemagglutinin and neuraminidase
- Thus, this induces mostly humoral immunity (anitbodies)
- However, this means that it is strain-specific, since there is lots of variation in the antigens between strains
- Regular update of vaccine composition is required
Describe live attenutated influenza vaccines (LAIV).
- Vaccine grown in embryonated chicken eggs
- It is not killed, just attenuated, so it can infect the upper airway in a limited manner, but not cause disease or spread
- It induces better immunity that the inactivated vaccine because it is live and can cause infection
- Induces humoral and cellular immunity
- Immunity induced is strain-specific, so it requires frequent updating
Describe how vaccines against influenza are created.
This is done by genetic reassortment:
- A chicken embryo is co-infected by a virulent influenza virus that is predicted to be the major strain next winter and a non-virulent donor virus
- Of the 256 possible resulting genotypes, ones are selected that have the genes that encode the virulent antigens, but the rest of the genes come from the donor virus
- Selecting the right genotype can be difficult
Describe how the process of creating vaccines against influenza can be simplified.
- Vaccine development usually requires genetic reassortment, after which the right genotype must be selected so that there are the virulent antigens, but not the rest of the virulent genome
- This genotype selection process is difficult, but it can be simplified by reverse genetics
- This involves the cloning of individual RNA segments for just the virulent antigens and non-virulent other genes into plasmids
- These can be inserted into the host cell, ensuring that the resulting vaccine has all the right proteins
Who decides which influenza strain is vaccinated against each year?
WHO, around February of that year
Can a universal vaccine against influenza be developed?
There are efforts, which aim to target highly conserved regions, such as the stalk of the haemagglutinin or neuraminidase or ectoderm of the M2 ion channel.
What is acute hepatitis?
Inflammation of the liver.
Describe the symptoms of acute hepatitis infection.
- Non-specific symptoms at first
- Generally feel off color
- Nausea and vomiting
- Hepatomegaly
- Jaundice
- Pale stool/dark urine
What are some biochemical changes during acute hepatitis infection?
After a short period of lag:
- First, there is an increase in alanine aminotransferase (ALT) -> Derived from hepatocytes and therefore a marker of hepatocyte injury.
- Then there is an increase in bilirubin and alkaline phosphatase (biliary)
- In severe disease, there may also be decreased synthetic activity of the liver (e.g. albumin synthesis)
Describe the pathogenesis of acute hepatitis.
- Virus itself may be relatively non-cytopathic (i.e. it does not directly cause cell death)
- Most changes are due to host respones:
- Includes lymphocytes, polymorphs and macrophages
- Cell-mediated immune response -> Cytotoxic T-cells kill cells and there is release of mediators like IFNγ, TNFα that are important in inflammation
- There is killing of bystander cells in addition to the infected cells
- The damage is relatively non-specific and common to various viruses
Which part of human immunity is important in hepatitis pathogenesis?
[IMPORTANT]
Cell-mediated immunity -> This is leads to bystander cell death and therefore damage to the liver, as well as inflammation.
What is the major cause of host cell death in hepatitis infections?
[IMPORTANT]
Cytotoxic T-cells
What are the different viruses and other causes that can lead to hepatitis (liver inflammation)?
Therefore, it is important to note that hepatitis is not only caused by hepatitis viruses.
How are hepatitis viruses written in shorthand?
HAV, HBV, HCV, HDV, etc.
These correspond to hepatitis A, hepatitis B, etc.
What are the type of hepatitis virus mentioned in the spec?
- Hepatitis B (HBV)
- Hepatitis C (HCV)
Summarise hepatitis A in terms of:
- Transmission
- Virus type
- Symptoms
- Diagnosis
- Vaccine
[EXTRA?]
- Transmission -> Feco-oral route
- Virus type -> Picornavirus
- Symptoms -> Mild and transient
- Diagnosis -> Detecting Hepatitis A IgM
- Vaccine -> Effective vaccine
Summarise hepatitis E in terms of:
- Transmission
- Number of cases
- Symptoms
- Diagnosis
- Vaccine
[EXTRA?]
What are the main differences between hepatitis virus types?
Quite distinct genetically and have different routes of transmission, but share “hepatotropism” (i.e. they all go to the liver)
What is the difference between acute and chronic hepatitis?
- Hepatitis is acute if it resolves within six months, and chronic if it lasts longer than six months.
- Acute hepatitis can resolve on its own, progress to chronic hepatitis, or (rarely) result in acute liver failure.
What are the main causes of chronic hepatitis?
Chronic hepatitis tends to be characterised by an imbalance between the pro-inflammatory drive (e.g. caused by alcohol) and natural tolerance mechanisms.
i.e. Increased inflammation + Reduced tolerance = Sustained inflammation
Compare the main viral causes of acute and chronic hepatitis.
- Acute -> Hepatitis A, B, C and E mostly, plus some other types like Yellow Fever
- Chronic -> Hepatitic B and C mostly
Describe the genetic material in hepatitis B and what Baltimore group it is.
Group VII:
- Have (partly) double-stranded DNA
- Use RNA polymerase to produce mRNA. When this mRNA enters cells, reverse transcriptase is used to produce double-stranded DNA. This is then transcribed into mRNA.
Describe the replication cycle of hepatitis B (HBV).
- Hepatitis B enters the host cell via endocytosis and uncoats, releasing its genetic material (in the form of rcDNA - relaxed circular DNA)
- cccDNA (covalently closed circular DNA) is formed in the nucleus from the rcDNA -> This is a source of new virions
- This can remain in the nucleus for a long time, so it makes it hard to get rid of the virus
- The cccDNA is trancribed, producing mRNA that can be translated to give viral proteins
- It also produces RNA that can be combined with the viral proteins to produce RNA-containing virions
- This RNA is then converted into DNA by reverse transcriptase, finishing the production of the new virion
How can hepatitis B’s replication cycle be targeted clinically?
Reverse transcriptase is required to convert RNA into DNA in newly-synthesised virions, meaning that RT inhibitors can be used as antiviral drugs.
How long is the incubation period for hepatitis B?
2-6 months
Describe the main routes of transmission for hepatitis B and C.
[IMPORTANT]
Blood, semen, or other body fluids -> Sex, sharing needles, or mother to baby at birth.
(Mostly via blood for HCV)
What are the two possible outcomes of hepatitis B infection in a person who is not vaccinated?
- Resolution (95% of cases)
- Chronic carriage (5% of cases)
Describe resolution of a hepatitis B infection.
Immunoglobulins are produced after the incubation period:
- First IgM increases, but then the levels fall
- Then IgG increases and remains high
Some HBV DNA may remain in the liver at very low levels.
Describe chronic carriage following a hepatitis B infection.
This occurs when the body does not produce a sufficient immune response against the virus, so it remains in the body chronically.
Draw the structure of hepatitis B.
[EXTRA?]
What are the two types of chronic carriers of hepatitis B?
All carriers have HBs antigens , but there are:
- High level carriers -> Have no antibodies against HBe, so they are HBe positive
- Low level carriers -> Have antibodies against HBe, so they are HBe negative
The high level carriers are at high risk of progression and cancer, due to continued T cell recruitment, as well as transmission.
Draw the genome of hepatitis C.
[EXTRA]
- The core elements that make up the virus itself are on the left
- The rest of the genome is non-structural proteins that are involved in replication of the virus
Compare the diversity of hepatitis C and HIV.
HCV has a higher diversity, which is because it has been within the human population for a longer time.
What role does viral escape play in outcomes of HCV infection?
- In patients who present with acute HCV infection, the virus will evolve under selection pressure from the immune response.
- The likelihood of clearance of the virus, and therefore resolution of the infection, is partly dependent on how much the virus evolves during the infection.
- During infection, there are periods of time where different antibodies are produced against the different emerging strains, and later there might be more broad cross-reactive antibodies.
Show the locations of action of the main drugs used to treat hepatitis C infection.
[EXTRA?]
- 1st Wave = Protease inhibitors (-previrs) -> Act on NS3
- 2nd Wave = Polymerase inhibitors (-buvirs) -> Act on NS5B
- 3rd Wave = NS5A inhibitors (-asvirs)
All three must be used together, or there is a high risk of viral escape.
What are the different herpes virus families and what sort of infection do they cause?
What are the different types of human herpes viruses?
Note: Only HHV-4 (EBV) is on the right of the spec. HHV 1,2,3 and 5 are right-hand side content.
Summarise herpes simplex (HHV-1 and HHV-2) infections.
[EXTRA]
- HHV-1 and HHV-2 are very common and tend to be what we think of when herpes is mentioned.
- HHV-1 infects primarily the lip area, establishing latency in the trigeminal ganglion. It has a 60% prevalence in the US.
- HHV-2 (genita herpes) infects primarily the ano-genital region through sexual transmission, establishing latency in the sacral ganglion. It has a 20% prevalence in the US.
How it establishes latency:
- There is lytic replication in the epithelial cells
- The virus then enters neuronal terminals
- The viruses are transported along the axons
- Latency is established in the nucleus of the neuron
- A reactivation stimulus may cause replication and anterograde transport
What are some complications of HHV-1 and HHV-2 infections?
[EXTRA]
Summarise HHV-3 (Varicella-Zoster virus) infection.
[EXTRA]
What does HHV-3 recurrence involve?
[EXTRA]
Summarise the primary infection by EBV.
- EBV infects the pharyngeal epithelial cells, which undergo lysis
- The EBV then infects naive and memory B cells
- EVC mimicks the signals that trigger the activation and proliferation of naive and memory B cells
- This proliferation is balanced by the T-cell immune response that kills cells since they present viral peptides on the surface
- The circular genome of the virus means that there is a latent infection of memory B cells
Summarise the emergence of EBV from latency.
- EBV can emerge from latency due to presence of the antigen that the B cell is specific to, but the virus itself can drive the process too
- Either way, the host B cell differentiates to become a plasma cell, while also regenerating the memory B cell population
- Differentiation to become a plasma cell triggers the virus to return to its lytic form, so it replicates and the viruses are released
- The viruses infect epithelial cells and new B cells, but the CD8+ cells are primed this time and are likely to stop the spread easily
Describe the different genes that are expressed at the different levels of EBV latency.
[EXTRA]
Summarise the roles of the different products of EBV and why they are important.
[EXTRA]
Give some experimental evidence relating to the herpes symbiosis.
[EXTRA]
(Barton, 2007):
- Mice were infected with herpes and then later infected with Listeria
- If the Listeria infection came several weeks later, then the herpes group performed better in terms of survival than the control group
- This suggested that the herpes could be a symbiont that improves survival
(Roberts, 2010):
- Elderly patients with anti-CMV (anti-herpes) antibodies were shown to have worse survival during the following years than the group without the antibodies
- This suggested that herpes was not a symbiont and it instead reduces survival
What is aciclovir used for and what is its mechanism of action?
- Used to treat herpes infections (HHV-1, 3 and 5)
- Acts by being converted into a triphosphate that acts as a chain terminator in DNA replication
What is infectious mononucleosis?
[IMPORTANT]
- Glandular fever
- It is caused by EBV (human herpes virus 4), which is spread by saliva
- It causes:
- Extreme tiredness
- Swollen glands in your neck
- High temperature
What are the main human tumour viruses?
What is a genetic change associated with Burkitt lymphoma?
- A reciprocal translocation between chromosomes 8 and 14 is characteristic of Burkitt lymphomas
- This translocation is common in this particular lymphoma because the virus opens up the region on chromosome 8 for transcription
- The translocation brings the myc gene (responsible for stimulating cell proliferation) next to an IgH gene for an immunoglobulin heavy chain
- Now the myc gene is under the control of the IgH enhancer and is therefore over-expressed, leading to rapid cell proliferation
- This leads to tumours
Summarise papilloma viruses.
[EXTRA]
Summarise the genome of HPV.
- In basal cells of the skin, the ‘early’ promoter (P97) is active -> This leads to transcription of the E1-E7 (e for early) proteins, some of which are involved in driving the cell cycle
- In epithelial cells, the ‘late’ promoter (P670) is active -> This leads to transcription of the L1-L2 (l for late) structural proteins
How is HPV infection linked to the differentiation of keratinocytes?
As the host cell passes through different levels of differentiation, the virus expresses different genes.
How is cell proliferation controlled in humans?
When a mitogenic signal is received, two pathways are triggered:
- CDK inhibits the RB1 that inhibits the cell cycle -> Thus, proliferation is stimulated
- p53 stimulates apoptosis and inhibits the CDK pathway via p21
When there is survival signalling, the p53 pathway is downregulated, so apoptosis does not occur and instead proliferation happens.
How does HPV cause benign tumours (i.e. warts)?
[EXTRA]
The HPV proteins interact with the cell cycle control proteins, causing proliferation to be favoured over apoptosis.
Give some experimental evidence relating to HIV and the Nobel prize.
The 2008 Nobel prize was won for elucidating that HIV was a retrovirus:
- Found that the ctopathic effect of the infection was the fusion of lymphocytes with each other into giant cells
- Used electron microscopy to isolate the virus particles and found that they contained the distinctive core of retro- and especially lentiviruses
- They were then able to demonstrate the action of the reverse transcriptase enzyme during HIV infection, which indicates a retrovirus
Summarise the different groups of HIV-1.
Describe the genome of HIV.
[EXTRA]
It is a single-stranded RNA with 3 reading frames that contain 3 main open reading frames (gag, pol and env), as shown in the diagram. It encodes 16 proteins.
Describe the structure of HIV-1.
- Outer lipid envelope with glycoproteins (gp120 and gp41)
- Matrix just within this, made of repeating units of P17 (encoded by the gag ORF)
- Two identical strands of positive single-strand RNA within a capsid (made of repeating P24 units)
State the main open reading frames (ORFs) in HIV-1 that encode structural proteins. What does each encode?
State the main open reading frames (ORFs) in HIV-1 that encode regulatory and virulence proteins. What does each encode?
Summarise the HIV-1 replication cycle.
- The virus attaches to:
- CD4 and CCR5 on T cells and macrophages
- CD4 and CXCR4 on naive T cells
- Viral core enters the cell via fusion of the viral and plasma membranes.
- Viral RNA is reverse transcribed to give DNA
- The viral core arrives at the nuclear pore and the genome enters the nucleus
- The proviral DNA is integrated into the host DNA
- In activated cells, the DNA is transcribed to give spliced and unspliced mRNA
- Viral proteins and genome are assembled
- New viruses bud out of the cell and are released
Summarise how HIV-1 enters cells.
- HIV-1 has glycoproteins in the envelope that enable binding and entry: gp120 and gp41
- gp120 binds to CD4 on T cells and macrophages
- This triggers a conformational change in gp120 that enables it to bind to CCR5 (on T cells and macrophages) or CXCR4 (on naive T cells)
- This in turn leads to even more conformational changes that allow the gp41 to extend out in an alpha helical arrangement -> They ‘harpoons’ the plasma membrane, causing pore formation
- The capsid can enter the cell due to membrane fusion
Describe the tropism of HIV-1 and why this is the case.
- CCR5 (R5) viruses are the transmitted type of HIV-1
- They can enter activated/memory CD4+ T cells and also macrophages/dendritic cells since they present CD4 and CCR5
- However, CXCR4 (X4) viruses evolve in ~50% of infected people, where they gain the ability to enter naive CD4+ T cells also, since they present CD4 and CXCR4
- This typically happens in individuals who progress to AIDS
Describe some HIV-1 virulence factors that antagonise the action of host cell restriction factors that aim to prevent HIV infection.
- SERINC5 inhibits entry into cells, but it is inhibited by Nef
- APOBEC3G inhibits reverse transcription, but it is inhibited by Vif
- Tetherin inhibits release of new viruses, but it is inhibited by Vpu
Summarise how HIV-1 spreads between host cells.
It can go by two methods:
- Cell-free spread -> Where new viruses are released and diffuse randomly new new host cells
- Cell to cell spread across a virological synapse -> This involves a transient adhesive junctions between cells that enable direct viral spread between them
Describe how viral load and CD4+ T-cells change over the course of a HIV infection?
Describe the two extremes of HIV progression to AIDS.
What is a good model for studying HIV in animals?
The SIV infection in macaque monkeys has a very similar immuno-pathogenesis to HIV, except that AIDS is reached usually within 2 years, as opposed to 10. This makes it a good model for study.
What is unusual about the natural infection of wild monkeys with SIV (similar to HIV)?
- In some cases, in spite of viral load remaining high, there is no progression to AIDS and the CD4+ T-cell population recovers
- This suggests that viral load does not correlate with progression to AIDS, but instead tbe primary correlate is non-specific systemic lymphocyte activation and cell death
- A related correlate is protection of the gut epithelial barrier from long-term damage
Describe how the gut is affected by HIV-1 infection.
- Normally, the gut has commensal flora in the lumen, surrounded by an intact epithelial layer that has GALT (Gut-associated lymphoid tissue)
- In HIV infection, the inflammation can breakdown the epithelial layer (physical barrier) and also causes helper T-cell death (immune barrier)
- Therefore, local immune homeostasis is not maintained, since bacteria can cross the barriers more easily
Summarise the vicious cycle that happens during HIV-1 infection.
Note: The main driving force for T-cell death in HIV infection is activation-induced lymphocyte death, which occurs when bacterial products translocate across the gut epithelium and activate immune cells in a non-specific way. The lymphocytes are programmed to apoptose after this.
Describe how latency of HIV works.
- HIV-1 can infect macrophages and T-cells (which can become memory T cells)
- When these cells are in long-term reservoirs, they are not active and therefore there is no transcription of the viral genome -> Thus, there is no immune response against the viral proteins and the virus is latent
- These viruses in these cells are resistant to drugs and the immune system
What are some potential approaches to curing HIV?
The problem with HIV is that a latent infection is very difficult to treat. The approaches include:
- ‘Berlin’ and ‘London’ patients, who received bone marrow transplants from individuals with no CCR5 receptors on their cells -> This resulted in there being no detectable virus for many years, making these patients essentially cured. However, this is not widely viable for every HIV patient.
- ‘Kick and kill’ approach, where latency is reversed by activating transcription in infected memory T-cells (so that they can be killed by cytotoxic T-cells) and then re-infection of new cells is prevented using strong anti-retroviral drugs
Describe the main events that happen after transmission of HIV-1.
- The virus crosses the genital or rectal epithelium through gaps in the epithelium or via dendritic cells
- Infected dendritic cells and T-helper cells take the virus to lymph nodes
- These lymph nodes distribute infected cells to all parts of the body
Describe the immune response to HIV infection.
- T-helper cells and plasma dendritic cells are recruited to the site of infection at the genital/rectal epithelium
- These can be infected but they also release chemokines and cytokines, which lead to recruitment of more cells
- Myeloid dendritic cells take up viral antigens arnd present them to helper T cells
- If the dendritic cell is infected, the antigens are also presented to cytotoxic T cells
- This leads to activation and clonal proliferation of helper and cytotoxic T cells
- Helper T cells help cytotoxic cell and B cell activity
Describe the early innate immune responses to HIV-1 infection.
Describe T cell responses to HIV.
Note: CTL stands for cytotoxic T cells.
Describe how the levels of different antibodies change throughout HIV-1 infection.
Non-neutralising antibodies form first:
- IgM rises at first, but it quickyl replaced by IgG that has higher affinity
- This is also followed by IgA (also at mucosa)
- They bind internal viral proteins, and are therefore not very effective, but are useful for HIV diagnosis
Neutralising antibodies:
- It can take many months for neutralising antibodies to form and they are generally specific only to the virus present at the time
- They are more effective at stopping spread, since they bind envelope proteins
- In about 20% of individuals, broadly-neutralising antibodies appear after years, and can neutralise a wider range of HIV strains
Explain the main experimental vaccine approaches for creating a HIV vaccine.
- Recombinant subunit vaccines
- Used to stimulate ANTIBODY responses
- Genes for the HIV envelope glycoproteins are transfected into a cell that expresses them -> This induces a response to them
- Vectored vaccines
- Used to stimulate CYTOTOXIC T CELL responses
- Genes for internal HIV proteins are cloned into an adenovirus vector that expresses them -> This induces a response in cytotoxic T cells
- Nucleic acid vaccines
- Used to stimulate ANTIBODY and CYTOTOXIC T CELL responses
- HIV genes are cloned into a plasmid DNA vector or RNA vector, and the genetic material itself produces an immune response (Check this!
- Conventional inactivated vaccines do not work for HIV-1 • Attenuated vaccines are too risky as may revert to virulence
Summarise the basis on which vaccines for HIV that target cytotoxic T cells work.
Uses vector-driven expression of HIV-1 genes to elicit cytoplasmic antigen:
- Viral vectors including poxvirus and adenovirus
- Nucleic acid DNA and RNA vector-based vaccines
What are some challenges of HIV vaccines targetting cytotoxic T cells?
[EXTRA]
What are some challenges of HIV vaccines targetting cytotoxic T cells and antibody production (combined)?
[EXTRA]
Summarise the effectiveness of HIV vaccines that aim to produce antibody responsess
They appear to show the most promise of the various vaccine types.
What are the main epitopes of HIV-1 that are targetted by broadly neutralising antibodies?
How can various stages of the HIV life cycle be targetted therapeutically?
Describe the structure and function of HIV reverse transcriptase.
- Heterodimer of p51 and p66 subunits with 2 enzyme functions: reverse transcriptase and RNAse H
- RNA template enters between ‘thumb’ and ‘fingers’ of enzyme and is reverse transcribed into DNA
- RNAse H destroys RNA template after new DNA strand is synthesised
Describe the function of DNA integrase in HIV and how this can be targeted by anti-viral drugs.
[IMPORTANT]
- Integrase engages both ends of viral DNA, cleaving host cell DNA and subsequently splicing in proviral DNA
- Inhibitors prevent strand transfer of viral DNA into host cell chromatin
Describe the function of protease in HIV.
[IMPORTANT]
- It is an dimeric aspartyl protease
- Cleaves immature precursor Gag/Pol polypeptides into mature functional peptides
- Inhibition with small-molecule pseudosubstrate prevents maturation of virions into an infectious form
Summarise the types of therapy and prophylaxis used for HIV.
Draw the structure of Covid-19.
Describe the genome of Covid-19.
- The genome is made of postive single-strand RNA so it can be translated directly
- It has 13 genes that are translated into a polyprotein
- There are two proteases encoded, that in turn cleave the polyprotein into the other proteins
Describe the infectious cycle of Covid-19.
- Virus binds to ACE2 on the surface of various human cells using spike proteins. This leads to internalisation via an endosome or fusion with the membrane (fusion involves a host membrane protease cleaving the spike protein to expose a fusion peptide).
- Viral genome is released.
- Host ribsosomes translate the genome (positive single-strand RNA) into the viral polyprotein
- Polyprotein is then cleaved by proteases into the individual enzymes and proteins required for replication
- Replication of the viral genome occurs using the RNA-dependent RNA polymerase to give genomic and sub-genomic (partial) RNA
- Transcription and replication of this genomic RNA occurs
- The subgenomic RNA is used to produce viral proteins in the endoplasmic reticulum
- The viruses are assembled
- The virus matures (undergoes cleavage so that it is infectious)
- The virus is released from the vesicle it is carried in
11.
Summarise how Covid-19 enters host cells.
- The virus binds to ACE2 on the surface of various human cells using the S1 domain of spike proteins.
- This leads to internalisation via an endosome or fusion with the membrane
- In order for fusion to happen, the transmembrane serine protease cleaves the S1 domain out, exposing the fusion peptide on the S2 domain
- This brings the virus closer to the membrane, leading to fusion of the two membranes
Give some experimental evidence for the entry of Covid-19 into cells.
- When HeLa cells that do not usually present ACE2 are made to present it, the infectious susceptibility of the HeLa cells increased.
- This was, however, not the case with mouse ACE2, suggesting it is not a great model for studying Covid-19.
Draw a timeline for a Covid-19 infection, including symptoms, etc.
Describe the immunopathology of Covid-19 in the lungs.
- Covi-19 primarily infects the epithelial cells in the alveoli, but also the type 2 penumocytes and any macrophages present
- Infection attracts macrophages
- These macrophages secrete cytokines that attract more leukocytes, which in turn release even more cytokines -> This leads to a cytokine storm that cause inflammation of the lungs
- Scar tissue can form in the lungs due to the deposition of fibrin
- Weakened blood vessels allow fluid to seep in and fill the alveoli, decreasing gas exchange
What are some markers of the systemic immunopathology of Covid-19?
There are a number of phenomena seen in both mild and severe disease:
- Decreased levels of lymphocytes (lymphopenia)
- This leads to increased T cell activation
- But this leads to exhaustion of the T cells, so there is lymphocyte dysfunction
- Neutrophils are increased, but other granulocytes and monocytes are decreased
- Increased production of cytokines and antibodies
What is ISG15 and what is its role in the immune response?
ISG15 = Interferon-stimulated gene 15
- This a protein produced in cells upon stimulation by interferons
- ISG15 acts to post-translationally modify host cell proteins in a manner that slows and inhibits the viral cell cycle (ISGylation)
- Some mechanisms:
- Increasing autophagic clearance of viral proteins
- Modifiying host proteins used in viral budding and release
How is ISG15 affected in Covid-19 infection?
- ISG15 levels rise as would be normally expected during an infection.
- However, coronaviruses have proteases that can remove the ISGylation of various host proteins
- So the levels of ISGylated proteins are low compared to other infections
How does Covid-19 affect antigen presentation by host cells?
It downregulates MHC-I and MHC-II.
How does Covid-19 affect the immune response in macrophages?
It leads to a shift to a pro-inflammatory state by increase pro-inflmmatory cytokines and decreasing anti-inflammatory cytokines.
Summarise the main vaccine approaches to Covid-19.
Adenovirus vaccines:
- An adenovirus is genetically engineered to contain the DNA for the Covid-19 spike protein
- It inserts this DNA into the human genome, so the cells can produce the spike protein and an immune response can be mounted
mRNA vaccines:
- mRNA encoding the Covid-19 spike protein is contained within a lipid nanoparticle
- This allows the mRNA to get inside cells, so the cells can produce the Covid-19 spike protein and an immune response can be mounted
In other words, both vaccines try to get the body to produce the spike protein, just in different ways.
