Flu, LCMV and HBV Flashcards
Pathogenesis
HPAI
Pathogenesis in past pandemics.
Epidemiology
Key stage: acquiring human-to-human transmission.
Control
Drugs
Vaccines
Evolution
Potential for pandemics
Evolution in endemic strains.
Classification of flu
Flu viruses classified by which HA and NA they have.
HA molecules
Single HA polypeptide precursor – HA0 - is cleaved by host proteases into subunits
HPAI cleavage site
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.
LPAI cleavage site
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.
MBCS and H3 strain.
Acquisition of MBCS in H3N2 in ferrets did not increase pathogenicity: other stuff is needed.
HPAI and mammalian pathogenicity
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.
Pathogenesis of past influenza pandemics.
Determined by virus binding, fusion and entry, by transcription and replication capacity, by modulation of the innate immune response, and by virulence release.
Why has HPAI not caused human pandemics?
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.
How likely is it that HPAIs will acquire human-to-human transmission?
5 mutations needed.
Number of mutations in environment
Difficulties in predicting.
Changes needed for HPAI to gain human-to-human transmission.
Adapt to replication at lower temp.
Adapt to human receptors.
Increase stability.
Adapting to replication at a lower temp.
2 substitutions needed to enhance transcription.
Adapting to human receptors.
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.
Increasing stability
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.
Mutations in environment.
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.
Study of acquisition of human-to-human transmission.
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.
Difficulties in predicting probability of HPAI causing human pandemic.
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.
Preparation for pandemics.
Surveillance of viruses that have acquired some of these mutations. If a 3rd acquired, consider culling and/or vaccine development.
Vaccines
Rapid evolution, but potentially predictable.
Difficult to stock-pile for HPAI pandemic, as we don’t know strain.
Common classes of flu chemotherapies
M2 protein inhibitors
Neuraminidase inhibitors
New antiviral strategies.
Targeting binding. Novel M2 and neuraminidase inhibitors. HA inhibitors. Rdrp inhibitors Targetting nucleoproteins or host functions.
M2 protein inhibitors
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.
Adamantane derivatives
M2 protein inhibitors. Amantadine, rimantidine.
Neuraminidase inhibitors - examples
Zanamivir, oseltamivir
Neuraminidase inhibitors - general.
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.
HA
Bind host receptors
Clathrin independent endocytosis
Undergo conformational change in low pH endosomes.
HA inhibitors
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.
Novel M2 channel blockers
o Further di-, tri- and tetrazole derivatives of adamantine have better antiviral activity against resistant strains.
o Some development of unrelated small molecule inhibitors.
Novel neuraminidase inhibitors
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
Rdrp inhibitors
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.
Inhibiting cap-snatching
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.
Targeting nucleoproteins
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.
Targeting host functions
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.
Evolution of MBCS
Proposed to evolve via addition of basic residues via strand slippage. H7 has acquired by recombination.
Pandemics in history
1918 – H1N1
1957 – H2N2
1968 – H3N2
(later H1N1 again).
The first virus vaccine
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.
Tracking evolution in real time.
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.
Antigenic cartography
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).
Disruption of antigenic map
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.
Choosing the vaccine strain.
The strain for the vaccine is chosen in February, and the vaccine season is October-November.
