Flu, LCMV and HBV

Question 1

Pathogenesis

Answer 1

HPAI

Pathogenesis in past pandemics.

Question 2

Epidemiology

Answer 2

Key stage: acquiring human-to-human transmission.

Question 3

Control

Answer 3

Drugs

Vaccines

Question 4

Evolution

Answer 4

Potential for pandemics

Evolution in endemic strains.

Question 5

Classification of flu

Answer 5

Flu viruses classified by which HA and NA they have.

Question 6

HA molecules

Answer 6

Single HA polypeptide precursor – HA0 - is cleaved by host proteases into subunits

Question 7

HPAI cleavage site

Answer 7

Both H5 and H7 have a multibasic cleavage site (MBCS) . Molecular studies clearly demonstrate that this is the basis of high pathogenicity because insertion of this basic cleavage site into low pathogenicity viruses such as H6 renders them high path. But not completely equivalent. Also, when inserted into an H3 strain, it did not cause increased pathogenicity – why?
o Molecular studies not yet shown why subtype specificity for HPAI is as it is.

Question 8

LPAI cleavage site

Answer 8

o LPAI cleavage site can only be cleaved by trypsin-like proteases in the respiratory tract and intestine: the MBCS is needed for systemic infections.

Question 9

MBCS and H3 strain.

Answer 9

Acquisition of MBCS in H3N2 in ferrets did not increase pathogenicity: other stuff is needed.

Question 10

HPAI and mammalian pathogenicity

Answer 10

o Zoonotic infections of H5N1 have about 60% fatality rate.
o In primate model, no evidence of replication outside the respiratory tract, but severe lung damage occurred. Alveolar damage leads to multiple organ dysfunction syndrome. Alveolar membrane damage also leads to lungs filling with fluid leading to respiratory distress. Zoonotic cases have a high fatality rate, with respiratory distress being the common cause of death.

Question 11

Pathogenesis of past influenza pandemics.

Answer 11

Determined by virus binding, fusion and entry, by transcription and replication capacity, by modulation of the innate immune response, and by virulence release.

Question 12

Why has HPAI not caused human pandemics?

Answer 12

Zoonotic infections have occurred in several subtypes: most do not proceed to pandemics due to a low R¬0 meaning that the virus is self-limiting. The problem is acquiring air transmission, meaning that sustained human to human transmission has yet to be seen.

Question 13

How likely is it that HPAIs will acquire human-to-human transmission?

Answer 13

5 mutations needed.
Number of mutations in environment
Difficulties in predicting.

Question 14

Changes needed for HPAI to gain human-to-human transmission.

Answer 14

Adapt to replication at lower temp.
Adapt to human receptors.
Increase stability.

Question 15

Adapting to replication at a lower temp.

Answer 15

2 substitutions needed to enhance transcription.

Question 16

Adapting to human receptors.

Answer 16

2 substitutions independently changed binding preference from α2,3-linked to α2,6-linked sialic acid receptors. A single mutation was still transmissible, but having both improved transmission. The loss of a glycosylation site improved binding, rather than the actual specific mutations.

Question 17

Increasing stability

Answer 17

Of binding and fusion. o Needs enhanced stability to prevent premature fusion. 1 mutation did this (H103Y). Stability increased both with respect to high temperatures and low pH.
Although this is most likely reason for selection of this mutant, the increased stability could be involved in stability of HA in aerosols, resistance to drought and etc.

Question 18

Mutations in environment.

Answer 18

5 mutations are needed for airborne transmission in ferrets, but many viruses already have 1, some have 2. 3 have been found in a bird.
• Deep sequencing needed to identify prevalence of these mutations.
• HPAI H7N9 can achieve airborne transmission within a few days of inoculation in a ferret, and could therefore potentially cause a pandemic.

Question 19

Study of acquisition of human-to-human transmission.

Answer 19

Use reverse genetics to insert mutations that have been epidemiologically identified as highly important (3) and do multiple passage through ferrets, naturally selecting for viruses that are adapted to mammals. When achieve ferret to ferret air-borne transmission, check for differences in genome: examine effects of mutations.

Question 20

Difficulties in predicting probability of HPAI causing human pandemic.

Answer 20

o Complex, since not only chance that mutations will occur, but also take into account positive and negative selection, the length of infection (longer  more time to acquire mutations in zoonotic infection), whether there are functionally equivalent mutations and etc. More research is needed to understand these parameters.
o Does each mutation give advantage, or do all have to be acquired to give advantage (hill-climb, or all-or-nothing).
o Chance could be decreased if
 Deleterious intermediate mutations are needed.
 Acquisition of mutants is order-dependent.

Question 21

Preparation for pandemics.

Answer 21

Surveillance of viruses that have acquired some of these mutations. If a 3rd acquired, consider culling and/or vaccine development.

Question 22

Vaccines

Answer 22

Rapid evolution, but potentially predictable.

Difficult to stock-pile for HPAI pandemic, as we don’t know strain.

Question 23

Common classes of flu chemotherapies

Answer 23

M2 protein inhibitors

Neuraminidase inhibitors

Question 24

New antiviral strategies.

Answer 24

Targeting binding. 
Novel M2 and neuraminidase inhibitors.
HA inhibitors.
Rdrp inhibitors
Targetting nucleoproteins or host functions.

Question 25

M2 protein inhibitors

Answer 25

o Adamantane derivatives. o M2 ion channels allow selective proton transport from endosome into viral envelope, triggering conformational rearrangements of HA and fusion. o Inhibitors block the ion channel pore. o Rapid emergence of drug resistance, as a single amino acid substitution is often sufficient.

Question 26

Adamantane derivatives

Answer 26

M2 protein inhibitors. Amantadine, rimantidine.

Question 27

Neuraminidase inhibitors - examples

Answer 27

Zanamivir, oseltamivir

Question 28

Neuraminidase inhibitors - general.

Answer 28

o Neuraminidase important to prevent aggregation on surface: needed to sialic acid which HA is bound to. Blocking NA limits cell to cell spread. o Structure of drug binding site well conserved between strains, so neuraminidase proteins generally have a wide spectrum of activity. o But a number of drug resistant mutations can arise given selective pressure, although these are rarer and take longer to acquire, usually, than for M2 protein inhibitors.

Question 29

HA

Answer 29

 Bind host receptors  Clathrin independent endocytosis  Undergo conformational change in low pH endosomes.

Question 30

HA inhibitors

Answer 30

 Synthetic receptor mimics.  Virus neutralizing antibodies - but very subtype specific, difficult to generate in large quantities etc. Potentially could bind either head, to prevent binding, or stem, to prevent conformational change.  Small molecules binding the stem region preventing conformational change. Several have been discovered, but tend to have a low resistance barrier, and be subtype specific, so not really pursued.  Arbidol stabilizes HA to prevent low pH transition to fusigenic state: licenced in Russia and China, but not elsewhere yet.

Question 31

Novel M2 channel blockers

Answer 31

o Further di-, tri- and tetrazole derivatives of adamantine have better antiviral activity against resistant strains. o Some development of unrelated small molecule inhibitors.

Question 32

Novel neuraminidase inhibitors

Answer 32

o Highly conserved structure, and slowly developing drug resistance means that it is a nice drug target. o Multimeric forms of zanamivir are more active. o Some new inhibitors in the pipeline:  Peramivir in phase III trials.  Other cyclopentane based compounds in development

Question 33

Rdrp inhibitors

Answer 33

o Elongates and capsnatches. Highly conserved, so good target. o Nucleoside analogues block elongation. Resistance seems slow to develop. Effective against strains resistant to current drugs. o Compounds to inhibit capsnatching. o Problem: substantial toxicity.

Question 34

Inhibiting cap-snatching

Answer 34

Cap analogues, cap oligonucleotides. Crystal structure of molecule has been used to predict subunit interfaces which disruption will be effective at, and predict molecular compounds that might inhibit this.

Question 35

Targeting nucleoproteins

Answer 35

o siRNAs decrease viral replication. o Molecules targeting oligomerization. by disruption of the salt bridges in the NP. o Induce aggregation of NP, exact mechanism unknown.

Question 36

Targeting host functions

Answer 36

o Can be dangerous as side effects  Sialidases prevent attachment by cleaving α2,6- and α2,3-linked sialic acids. Some have got as far as phase 2 clinical trials.  Inhibiting endocytosis or acidification. Problem: this is a key physiological function, would need to target somehow. Nonetheless, some analysis as to toxicity profiles and etc is going on.

Question 37

Evolution of MBCS

Answer 37

Proposed to evolve via addition of basic residues via strand slippage. H7 has acquired by recombination.

Question 38

Pandemics in history

Answer 38

1918 – H1N1 1957 – H2N2 1968 – H3N2 (later H1N1 again).

Question 39

The first virus vaccine

Answer 39

The first vaccine was in the 1940s: 70-80% effective for a few years, then no longer. Same happened with next vaccine. This is due to incremental change in the antigens presented by the virus.

Question 40

Tracking evolution in real time.

Answer 40

We know what is happening due to the WHO flu surveillance network: there are 5 centres around the world which are sent flu samples to be analysed. Ab based assays measure antigenic differences, and these are used for vaccine development. About 20000 viruses are isolated each year.

Question 41

Antigenic cartography

Answer 41

Used to measure similarity between strains. Both axes measure distance. A map usually has several clusters, and a strain from roughly the middle of the cluster is used for the vaccine, as it will protect against all the strains in that vaccine. Every 3 years (roughly: range is about 1-8) a virus jumps much further, creating a new cluster. A couple of cluster jumps, and the virus has escaped immunity (which is why we get flu roughly every 10 years).

Question 42

Disruption of antigenic map

Answer 42

The map can be disrupted (as in 2007) by changes relevant not to our immunity by to the HI assay – in 2007 variation in how well it bound turkey red blood cells occurred, but was corrected by switching to guinea pig red blood cells.

Question 43

Choosing the vaccine strain.

Answer 43

The strain for the vaccine is chosen in February, and the vaccine season is October-November.

Question 44

Flu evolution due to selection or drift?

Answer 44

When a virus jumped from humans to pigs, it ceased to be under immune pressure, because the pigs get eaten within 6 months, and new ones are raised, so there is a constant supply of naïve hosts. With this cessation of immune pressure the virus CEASED TO CHANGE ANTIGENICALLY. In other words, if a virus doesn’t have to change it won’t. Therefore, antigenic change is due to selection, and could therefore potentially be predictable.

Question 45

Analysing which mutations cause antigenic change.

Answer 45

Analysed which residues were changed in cluster transitions, then tried some site-specific mutagenesis. For some of these a single amino acid change lead to a cluster transition! For others, needed 2. The vast majority lead to no antigenic change. Gradual genetic evolution, but punctuated antigenic evolution. Clustered antigenic drift with similar antigenic distances between consecutive cluster centroids.

Question 46

Which mutations cause cluster transitioning?

Answer 46

There were only 7 residues that caused cluster transitioning, all around the receptor binding sites. Possibly it can change antigenically at other sites, but it doesn’t in nature. But these 7 sites should be the ones it wants to conserve! Therefore, the only Abs that drive selection of new strains must be the ones that bind to the receptor binding site. And it doesn’t simply drift antigenically because there is a constraint on mutation since there is a fitness cost to mutating here.

Question 47

What was originally thought to cause cluster transitioning?

Answer 47

thought to be 135, with at least 4 spread across different antigenic sites to cause a single transition.

Question 48

Results of genetic changes causing cluster transitions.

Answer 48

Almost all changes changed biophysical properties: charge, or volume Mutations that cause cluster change also reverse change if reversed.

Question 49

Predicting mutations.

Answer 49

Unpublished experiment – Make all single substitutions at the 7 positions (140 viruses), select the 20 that might be advantageous (e.g. exposed, so most have to be hydrophilic, so many possibilities out for that reason)  measure the binding site affinity  possibly we could predict the next strain.

Question 50

Highly pathogenic avian influenzas - classes

Answer 50

Avian influenzas H5 and H7 are highly pathogenic (HPAIs).

Question 51

Importance of LCMV

Answer 51

o LCMV because good model for HBV. Even though bisegmented single stranded ambisense genome rather than partially double stranded like HBV.

Question 52

Mechanisms of persistance (LCMV, HBV)

Answer 52

Important parts of immune system. | Avoiding immune system.

Question 53

Outcomes of persistent infection.

Answer 53

```  Immune complex disease (LCMV and HBV)  Autoimmune disease (LCMV)  Growth hormone deficiency (LCMV)  Thyroid dysfunction (LCMV)  Alterations in behaviour (LCMV)  Cancer ```

Question 54

Targeting LCMV; the immune system.

Answer 54

CTL, CD8, CD4, IFNy Long term memory cells.

Question 55

Targeting LCMV; evidence for importtance of CTL.

Answer 55

KO mice  dependent on perforin mechanism CTL for clearance of acute. CD8 major early defence, and early immunopathology.

Question 56

Targeting LCMV; importance of CD8, CDD4 and IFNy.

Answer 56

All needed to clear chronic. Even when virus cleared from blood, often viral genes detectable in organs.

Question 57

Targeting LCMV; memory cells

Answer 57

long term memory cells – central memory cells home to lymph nodes, and effector memory cells have tissue effects and reside there. In LCMV, TCM form, then on exposure to antigen differentiate to TEM.

Question 58

Importance of CTL in clearing HBV.

Answer 58

CTL response appears important in clearing acute infection, but chronic can be controlled without cell lysis, and many drugs used to treat it focus on Type 1 IFNs.

Question 59

Targeting LCMV and HBV. Superinfections.

Answer 59

Superinfections of LCMV clear HBV, possibly of HCV – suggests stimulation of antiviral agents important.

Question 60

LCMV and HBV. Avoiding the immune system

Answer 60

Evasion of induction. Blocking effector arm Modulation Overwhelming immune response.

Question 61

HBV, avoiding the immune system, evasion of induction.

Answer 61

Liver = immunoprivileged site. Little MHC I. Downregulation of MHC I Antigenic variation.

Question 62

Antigenic variation LCMV

Answer 62

o LCMV high level of mutation – little proof reading o Escape variants emerge in transgenic mice which avoid the TCR or Ab they express if viral load is high enough. If low, then cleared before escape variants develop. But, if change gradual, cross reactivity between sequential epitopes allows host to mount sequential immune responses.

Question 63

Escape mutant in HBV

Answer 63

not common, partly because it is a DNA virus, so has lower rates of mutation. Mutation may occur in RNA intermediate phase.

Question 64

LCMV and HBV: blocking effector arm.

Answer 64

* Decoy receptors * Resistance to lysis? * Resistance to cytokines?

Question 65

LCMV and HBV - modulation of the immune system.

Answer 65

INDUCTION OF TOLERANCE. o LCMV; if mice are transgenic for an LCMV specific TCR, negative selection means that these T cells are eliminated during development of central tolerance. o HBV: equivalent only occurs in utero as in humans this is when tolerance is established.

Question 66

CTL exhaustion

Answer 66

Well modelled in LCMV. Mechanism Reasons

Question 67

Reasons for CTL exhaustion (LCMV)

Answer 67

Because exhaustion and persistent infection better than continued inflammation and death due to inflammatory death to liver and lungs.

Question 68

Causes of CTL exhaustion (LCMV)

Answer 68

Defects in signalling Active suppression Induction by virus.

Question 69

Causes of CTL exhaustion (LCMV) - defects in signalling.

Answer 69

Defects in signalling and metabolism: metabolic deficiency --> exhaustion. Signalling e.g. downregulation of TRAF-1 --> poor co-stimulation.

Question 70

Causes of CTL exhaustion (LCMV) - active suppression.

Answer 70

FROM OTHER CELLS o IL-10 produciotn by APCs ( in HBV, CD4 cells that remain after viral suppression may also produce this). o Stimulation of inhibitory receptors PD-1 and LAG3. FROM WITHIN CELL o Transcriptional repressors e.g. Blimp1. o Loss of Tbet expression.

Question 71

Causes of CTL exhaustion (LCMV) - induction by virus

Answer 71

Rapid replication where T cells are causes exhaustion. • Isolates with T cell tropism due to sequence of viral glycoprotein increase exhaustion. • Isolates which are resistant to anti-replicative properties of type 1 IFNs are more likely to cause exhaustion. • Lots of antigen where there is little CD4 help may lead to cell death.

Question 72

CTL exhaustion in HBV.

Answer 72

* Virus appears to suppress specific CD4 cells which help CTLs: treat with antiviral and specific CD4s emerge. * acute rapidly cleared HBV patients have strongly virally specific CTL responses, while those with weak and exhausted ones tend to become chronically infected.

Question 73

Outcome of persistence, immune complex disease.

Answer 73

LCMV and HBV – lots of Ag --> poorly cleared --> trapped in small blood vessels --> immune damage --> glomerulonephritis, ateritis, choroiditis.

Question 74

Outcome of persistence, growth hormone deficiency.

Answer 74

LCMV • Replicates in GH producing cells. Sequestration of GH transactivator factor by nuclear protein  suboptimal expression of GH genes  GH deficiency.

Question 75

Outcomes of persistence, cancer.

Answer 75

chronic infection --> rapid cell turnover and mutagenesis --> DNA damage, chromosomal abnormalities, viral DNA damage, integration into the host genome and gene X activation --> loss of cellular growth control --> hepatocellular carcinoma.

Question 76

Emergence - general discussion

Answer 76

Newly vs re-emerging Changes in host Changes in environment Changes in pathogen.

Question 77

Emergence - key points

Answer 77

``` General discussion Reservoirs Contact Mutability and mutations Changes increasing chance of emergence. ```

Question 78

Reservoir for influenza

Answer 78

Birds

Question 79

Reservoir for paramyxos.

Answer 79

Bats. Hendra and Nipah.

Question 80

Reservoir for corona

Answer 80

Bats

Question 81

Reservoir for HIV

Answer 81

Primates.

Question 82

Contact in emerging virus infections.

Answer 82

Often more than one. E.g. HIV M,N,O,P all separate jumps

Question 83

Important mutations

Answer 83

Needs to be able to enter/replicate in cells. Needs to acquire ability to transmit (increase R_0). MERS SARS Influenza. Important for health impact but not emergence: virulence mutations.

Question 84

SARS ability to transmit.

Answer 84

 SARS was poor: combined with intensive scientific research and collaboration  contained. Also, low R0, and only infectious when symptomatic.

Question 85

MERS ability to transmit.

Answer 85

MERS R¬0 estimated around 0.6, so though can human-human transmit, will need to be more efficient -->pandemic.

Question 86

Changes increasing chance of emergence

Answer 86

Increased contact due to changes in host organisms and vectors, due to changes in secondary hosts, increased population pressures, increased human susceptibility.

Question 87

Changes increasing chance of emergence: increased contact due to changes in host organisms and vectors.

Answer 87

Climate change.

Question 88

Changes increasing chance of emergence: increased contact due to changes in secondary hosts

Answer 88

 Increased contact with primary hosts • hunting, eating • Pets (especially rise of more exotic pets, some of which are intermediary hosts being both more similar to humans , and highly susceptible) etc.  Agriculture - farmed near primary hosts, civet cats kept in cages near wild bats in live animal markets.  Urbanisation (pigs/birds in close proximity to humans).

Question 89

Changes increasing chance of emergence: increased contact due to increased population pressure.

Answer 89

Clearing of forests (Nipah), drought (Ebola) leads to increased contact.

Question 90

Changes increasing chance of emergence: increased human susceptibility.

Answer 90

Niche e.g. for poxvirus caused by absence of smallpox. Increasing numbers of naive-to-pox hosts --> potential for epidemic.

Question 91

Changes increasing emergence: increased chance of spread.

Answer 91

o Increased international travel – SARS in 2003 to 30 countries, international hubs important. o Population movements/war zones. Along trade routes, sex workers and HIV etc. o Technological changes: blood borne with blood transfusions, poor use of nebuliser in hospital in SARS epidemic etc etc.

Question 92

Re-emerging viral infections.

Answer 92

``` Altered vector/host presence Introduction to previously naive populations Spread due to technology Failure of control Bioterrorism ```

Question 93

Re-emerging viral infections: altered vector/host presence.

Answer 93

1) Climate change  Spread of midges, Blue tongue  Spread of mosquitoes, dengue: spread of serotypes so overlapping may lead to increased incidence of Dengue shock syndrome.  Alteration of bird migration (e.g. West Nile Virus lineage 2 spread in Europe). If potential vectors present, this could have effect. E.g. WNV wide vector range. 2) Global trade. 3) Population movements  The Hajj pilgrimage: crowded conditions, people from all over the world. Spread of polio from Nigerian outbreak Potential for spread of MERS?

Question 94

Re-emerging viral infections: introduction to previously naive populations.

Answer 94

o Measles/small pox to Americas | o Expanding reservoir – West Nile Virus. Non-specific host range. Increase in zoonoses.

Question 95

Re-emerging viral infections: spread due to technology.

Answer 95

Blood transfusions.

Question 96

Re-emerging viral infections: failure of control.

Answer 96

o Multi-drug resistance o Failure to vaccinate – polio Nigeria. o Social factors: war/natural disasters  failure to vaccinate, poor sanitation etc.

Question 97

Re-emerging viral infections: bioterrorism.

Answer 97

Not impossible to imagine deliberate release of a dangerous virus e.g. smallpox, or design of a virus with great virulence.

Question 98

Controlling emerging viral infections

Answer 98

Prevent jump. Improve surveillance and diagnosis. Re-emerging of infections. Stockpile drugs.

Question 99

Controlling emerging viral infections: preventing jump.

Answer 99

a. Alter behaviour – less wildlife cuisine, no xenotransplants. b. High risk animal viruses e.g. H5N1 with adaptations to mammalian host. Culling. c. Vaccinate reservoir – not normally an option, but being considered for MERS where the reservoir is the camel population and therefore both small and available to humans.

Question 100

Controlling emerging viral infections: improve surveillance and diagnosis.

Answer 100

a. This only really occurs where θ is low. So doesn’t work for HIV, but worked well for SARS.

Question 101

Controlling emerging viral infections: re-emerging infections.

Answer 101

a. Vaccinate (e.g. Saudi Arabia insisted on evidence of vaccination for polio when polio re-emergence happening in Nigeria). b. Isolate and contact tracing.

Question 102

Controlling emerging viral infections: stockpile drugs.

Answer 102

a. Difficult to develop if no model to test on. Most obvious with smallpox, where several drugs have been developed, but cannot be tested according to FDA guidelines because no cases of smallpox left. b. If jumps have only been zoonotic, difficult to develop in the first place. c. Difficult to predict which will make jump; cannot develop drugs to all potential emerging viruses as not economically viable.

Question 103

Very general emerging infections

Answer 103

Emerging Re-emerging Control.

Question 104

New antiviral strategies: targeting binding.

Answer 104

Convalescent immunoglobulin | sialidase.

Question 105

Example of sialidase in phase II clinical trials

Answer 105

DAS181, a bacterial sialidase, tagged to amphiregulin to give longer action and decreased spread to systemic circulation.

Question 106

Druggable targets in HIV-1

Answer 106

``` Entry inhibitors RT inhibitors Integrase inhibitors Protease inhibitors Maturation inhibitors. ```

Question 107

Causes of resistance

Answer 107

Inadequate dose of drug Inadequate penetration of drug Inefficient action of drug.

Question 108

Resistance to drugs

Answer 108

Is on a continuum.• Resistance is likely to be a cumulative effect. Viruses are usually considered resistant if there is detectable replication despite a normal therapeutic drug dose. Toxicity is the main limiting factor to successfully combating resistance.