Virology Flashcards
What is a virus?
Very small obligate intracellular parasite - non living.
Can have either single/double stranded RNA/DNA genomes
Capsid encoding organisms.
How were viruses first distinguished from other microorganisms?
1892 Ivanovsky ground up leaf tissue + filtered it -> filtered liquid had agent of disease not the concentrated filtrate
1898 Beijerinck repeated but said filterable agent was not a small bacterium
Electron microscope allowed for x100,000-fold magnification
What does the Baltimore classification scheme demonstrate?
7 genome types based on replication strategies - all must make mRNA that can be translated by host ribosomes.
DNA genomes - 2kb ssDNA Circovirus -> 2.8 Mb Pandoravirus
RNA genomes - 1.7kb -ssRNA hepatitis -> 31kb -ssRNA Coronavirus
Escape/progressive hypothesis for viral origins
Mobile genetic elements exited one cell + entered another via acquisition of structural protein.
Retrotransposons move via RNA intermediate like retrovirus.
Reduction/regressive hypothesis for viral origins
Viruses degenerate so retain genetic info for parasitic way of life -> loss of previously indispensable genes + reduction in genome size
- obligate intracellular parasites (Chlamydia + Rickettsia bacteria) evolved from free living ancestors, cant make ATP or proteins
- Mimivirus has relics of genes encoding tRNAs, aminoacyl tRNA synthetase + TFs so previously non-parasitic BUT evidence of horizontal gene transfer
Virus first hypothesis for viral origins
Independent entities evolved parallel/before cellular life from self-replicating mols in RNA world BUT all viruses need cellular host for replication
- 1st replicating mol had RNA not DNA, maybe circular ssRNA of ribozymes could infect first cells
- complex enveloped DNA virus became resident of emerging eukaryotic cell (endosymbiotic event)
What is the general structure of a virus?
Metastable structures (non-covalent bonding)
- extracellular virions stable to protect genome
- intracellular virion must open to release genomic contents
Watson + Crick 1956 EM studies showed rod vs spherical viruses - later helical & icosahedral symmetry.
TMV structure
Helical, +ssRNA 6.4 kB, single protein capsid
Each protein subunit binds 3 nucleotides + adjacent subunits, hollow helix w/ pore.
Parvovirus structure
60 subunits, very small (18-26nm) - 12 pentons/capsomers at vertices
3 subunits per face in head-head + tail-tail gives rotational symmetry.
T=3
Subunit proteins about 100kDa -> larger viruses need increased subunit number + increase triangulation value.
Triangulation values + icosahedral structure with examples
Always 12 pentamers but number of hexamers varies w/ size.
T=1, 20 faces + 60 subunits
T=3, 60 faces + 180 subunits
T=4, 80 faces + 240 subunits
- Nodamura virus (T=3), coat proteins defined by occupancy of structurally distinct environments.
- Brome mosaic virus (T=3), pentons + hexons composed of same subunit.
- Adenovirus (T=25), pentons + hexons composed of different subunit proteins.
What is the role of glycoproteins in membrane bound viruses?
Membrane from host cell but studded w/ glycoproteins in bilayer -> most are oligomers
e.g. influenza HA is trimeric
Glycoproteins act as receptors, antigenic determinants + mediators of cell fusion.
Examples of Membrane bound viruses
Measles (-ssRNA) helical sym, membrane has HA & fusion protein (F)
Herpes simplex (dsDNA) has icosahedral sym.
Influenza: 8 separate helical nucleocapsids interact w/ ribonuclear proteins -> organise each RNA into helix, further folding by viral P proteins (sequence specific) at 5’ & 3’ end.
Matrix protein holds structure together.
Has HA to bind respiratory epithelial cells + NA enzyme allows exit
What are the requirements for replication to occur in a cell?
Susceptible cells have functional receptor for given virus - may or may not support replication (HIV cant infect primate cells)
-> resistant cells have no receptor
Permissive cells can support replication.
What did Ellis & Delbruck develop in 1939?
Study of bacteriophages in E.coli. Multiplicity infection (10 phage:1 bacteria) - diluted to prevent further absorption.
Sample taken at intervals, virions counted using plaque assay
-> one step growth curve formulated
Avg burst size is 100 phage from 1 E.coli
What happens in the latent phase of a growth curve?
Eclipse - no viral particles detected, uncoating, viruses actively transcribed + replicating, protein synthesis starts
Intracellular accumulation - proteins + viral genome self-assemble into virions that accumulate in cytoplasm-> viruses CAN be detected
What happens in the rise period of a growth curve?
Viral particles accumulate to threshold level -> triggers lysis, virions released increasing extracellular phage conc rapidly.
BUT adenovirus bucks trend: membrane bound (needs to acquire membrane so we see extracellular virus before intracellular
Receptors for viral attachment
Protein receptors tissue specific - tropism receptors dictate host range
Carb receptors less specific - presence determines cells resistance (co-receptors can be required e.g. HIV)
Polio virus receptor (Pvr)
CD122 indentified by transfecting mouse cells w/ human cDNA library.
Polio is pseudo T=3, VP1/VP2/VP3 form subunits of capsid
-> 5 VP1 form 5-fold symmetry axis, penton has canyon in capsid which is recognition site for receptor
1 polio interacts w/ 60 receptors
How does Influenza virus attach to cells?
Via a carb receptor - HA binds -ve terminal sialic acid on surface glycoproteins
Human HA binds a2-6 sialic acid, Avian HA binds a2-3 sialic acid. Sialic acid ubiquitous.
Adhesion triggers entry across membranes -> genome injection, membrane fusion, endocytosis (dictated by whether virus enveloped or not).
Methods of entry by naked and enveloped viruses
Naked - genome injection (bacteriophages), endocytosis (adenovirus, polio)
Enveloped - plasma membrane fusion (Sendai, HIV), endocytosis followed by endosome membrane fusion (influenza)
Entry into cells via membrane fusion
Unique to membrane bound viruses.
e.g. Measles (RNA), Herpes (DNA)
Membranes fuse together emptying virion into cytoplasm - then uncoated to release nucleocapsid.
DNA must translocate to nucleus membrane via filaments to be uncoated.
Receptor mediated endocytosis
Used by both membrane bound + naked viruses.
- Viruses bind receptor + accumulate in clathrin coated pit in membrane.
- Pit forms enclave which is enclosed by dynamin -> clathrin coated vesicle
- virus uncoated + released into cytoplasm
- ligands bound to receptor remain in vesicle (pH~7.0)
- fuses w/ endosome
- protons added lowering pH (~6.0) + fuses w/ lysosome to be degraded
- receptors recycled
Define uncoating in viral replication
Releasing of viral genomic material for replication to occur.
Occurs simultaneously w/ entry in measles.
- DNA viruses complete uncoating at nuclear pore
- RNA viruses uncoat by fusing w/ plasma membrane/endocytic vesicle membrane -> releasing genome into cytoplasm
How does uncoating work at the plasma membrane?
Enveloped RNA only e.g. Paramyxoviridae - Sendai, Measles, Respiratory synctial.
- HA adheres to surface receptors
- Fusion of protein F engages + viral/host membrane fuse
- Viral nucleocapsid (-ssRNA) + viral proteins released into cytoplasm
- Synthesis of +sense mRNA occurs followed by translation
What are the details of Paramyxoviridae fusion?
F protein synthesised as F0 precursor - cleaved to F1 & F2 (connected by disulphide bond) by host cell protease.
- fusion peptide buried between F1 & F2 subunits
- Binding of HN causes conformational change exposing fusion peptide -> highly hydrophobic so embeds in host membrane
How does uncoating via the formation of a pore in the endosome work?
Polio binds to PVR (CD155) + conformational change occurs.
- Pocket lipid lost & hydrophobic N termini of VP1 + VP4 displaced to surface so inserts into endosome membrane
- Pore formed which +ssRNA genome bound w/ VPg protein passes through
How does uncoating of the Influenza virus take place? (Stage 1: fusion)
- HA1 binds receptors w/ sialic acid -> endocytosis.
- Import of H+ ions acidifies endosome causing HA conformational change, reveals fusion peptide in HA2
- Loop region in HA2 becomes coiled coil, fusion peptides reoriented towards endosome membrane
- Alpha helices pack down bringing 2 membranes closer together allowing fusion
How does uncoating of the Influenza virus take place? (Stage 2: release of RNA genome segments)
- M2 ion channel homotetramer, forms pore at low pH allowing protons to enter viral capsid
- drop in pH causes conformational change of M1-> breaks bond which tethers vRNP to M1
- M1-M1 bonds broken so capsid dissociates
- release of vRNP reveals nuclear location signals allowing nuclear import
How does uncoating in the cytoplasm by ribosomes work?
e.g. Semiliki Forest virus
- enters via clathrin-dependent endocytosis
- acidification of endosome triggers fusion of viral + endosome membranes
- viral nucleocapsid released into cytoplasm but still tethered to cytosolic endosome membrane
- ribosomes bind nucleocapsid + hydrolyse
- each ribosome binds 3-6 mols of C protein causing detachment
- ribosomes bind +ssRNA genome & translation begins (still tethered to membrane)
How does uncoating at the nuclear pore work?
e.g. stepwise in adenovirus
- integrin contacts fiber receptor bound to penton -> triggers clathrin mediated endocytosis
- endosome acidifies, fibres released from virus + penton bases drop off leaving only hexons
- protein VI causes endosome lysis, embeds in membrane destabilising it
- binds MTs in host to traffic to nuclear pore
- hexon proteins interact w/ histone proteins
- importin-7 + importin-B bind histone H1 -> import of protein into nucleus triggering capsid disassembly
Transportin + protein VII help DNA import
Why is nuclear import easier for parvoviruses & hepadnoviruses?
Very small so can just enter via nuclear pores.
What are the methods of leaving the host cell?
Naked viruses - cell lysis
Membrane bound - budding/ exocytosis as need to acquire a membrane from ER/golgi/plasma mem
How do naked viruses leave host cells?
Normally results in cell lysis:
Adenoviruses + Polio shut down production of cellular proteins.
- eIF4A cleaved by viral protease 2A (encoded P2) so subunit lost from IF complex + ribosome cannot bind
Polio alters membrane permeability (2Bpro tetramer forms pores in membrane)
Bacteriophage release cytolytic molecules.
- large quantities of virions accumulate prior to release
Describe how Polio induces lysis
Shuts down protein synthesis:
- eIF4F which binds 5’-7-methyl guanosine CAP is cleaved by viral protease 2A (encoded by P2)
- eIF4G subunit lost from IF complex preventing cellular RNA from binding ribosomes
Alters membrane permeability of host cells -> 2Bpro is tetramer of 99aa peptide, forms pores in membrane -> lysis
What does the Polio virus genome consist of?
Polio genome (T=3)
- P1 encodes capsid units
- P2 encodes proteins for interaction w/ the host (e.g. protease 2A)
- P3 encodes proteins which participate in genome replication
- IRES (internal ribosome entry site) 500bp allows ribosome binding + translation
Describe how Polio can induce its non-lytic release in the GI tract
- upon replication in polarised epithelial cells, only released from apical surface
- virions enclosed in autophagosome vesicles (formed by 2BC & 3A proteins from Golgi membranes) - happens during late infection
- fusion of vesicle w/ mem -> non-destructive from cell
Where do various enveloped viruses acquire their membrane from?
Exocytosis - via budding
Envelope acquired from plasma membrane (influenza) or internal membranes of secretory pathway (herpes)
Can (rhabdovirus, paramyxovirus, togavirus) or can not (retrovirus) cause cell death.
How does exocytosis take place for the influenza virus?
Acquires membrane from host cell membrane. Viral membrane studded w/ proteins: HA, NA + M2.
Proteins synthesised & delivered via secretory systems:
- synthesis + co-translational membrane insertion into ER
- glycosylation stars in rER + continues in Golgi - glycoproteins transported to membrane via vesicle
Virus w/ nucleocapsid migrates to virus modified membrane + buds off to form free infectious virus.
** matrix, capsid + replication enzymes synthesised by free ribosomes vs viral mem proteins translated on ribosomes associated w/ ER
How does assembly & exocytosis occur in Herpes virus?
2 cycles of envelopment.
- UL31 & UL34 bind lamina prteins when phosphorylated by US3 (kinase) -> catalyses disruption of nuclear lamina + promotes budding.
UL51 helps virus leave ER, nucleocapsid has associated tegument proteins -> amass around nucleocapsid + important for replication cycle. - glycoproteins (gE, gI, gM, gD) interact w/ Tegument proteins in trans Golgi network
- virus formed at Golgi + is exocytosed
**virus acquires nuclear & golgi membrane
Maturation of virus particles
-> when virus becomes infectious
Viral proteins need to proteolytically processed post-assembly.
Happens late in assembly (Polio) or following release of immature virions from host cell (retrovirus)
How are retrovirus particles rearranged after exocytosis?
Gag polyprotein layer beneath viral mem found in immature virus
-> cleaved by HIV protease -> infectious HIV
Protease used as drug target for HIV treatments.
DNA packing signals
e.g. Polyoma & adenoviruses use short sequence repeats close to origin
SV40 in regulatory region - has 6 tandem binding sites for viral TF sp1.
Bound sp1 interacts w/ capsid proteins + stimulates assembly
Adenovirus IVa2 proteins recognises packing signals.
RNA packing signals
e.g. HIV type 1 genome
signal-psi recognised nucleocapsid proteins - necessary but not sufficient for HIV packaging, only found in unspliced genomic RNA
-> binds DIS + part of DLS
TAR + poly A loops also required
Packing of segmented genomes
Each of 8 genome segments in Influenza has unique signal at both ends.
Segments arranged in virus in specific pattern -> interactions between the segments
What does virion assembly depend on?
Concentration - components concentrated in ‘factories’ -> internal membranes can be sites of assembly providing means of concentrating proteins
Can be either independent or dependent on host machinery.
Host dependent assembly
- chaperones catalyse/assist folding of individual proteins + assembly of capsid/nucleocapsid
- viral proteins + nucleic acids from sites of assembly
- host secretory pathways processes + moves viral particles
- host nuclear import/export machinery moves viral nuclear proteins + nucleic acid in & out of nucleus
What are the types of addresses embedded in amino acid sequences?
Signal - target correct membranes
Retention - remain in appropriate membranes
Nuclear localisation - go to nucleus
Nuclear export - ensure viral mRNA/ribonuclear proteins moved into cytoplasm
Components can travel short (across membrane) or long (to site of replication, or assembly) distances across the cell.
How are protein shells assembled in viruses? (give examples)
Self assembly:
- from individual protein mols
e.g. Simian virus, need large excess of VP1 to VP2/3 (stoichiometry) for spontaneous formation
-from polyprotein precursor
e.g. Polio, gets around need for high conc. -> VP1-4 joined together, protease then cleaves bonds between subunits except VP1-4, 2 & 4 not cleaved until whole virus assembled w/ RNA genome
Assisted assembly: uses chaperones
e.g. Adenovirus, 3 protein IIs generate hexon timer, requires 100kDa L4 chaperone host cell protein
Sequential assembly (in bacteriophage T4)
Genome inserted into pre-formed protein shell e.g. Herpes, Adenovirus
In bacteriophage T4:
It ensures orderly formation of viral particles + virion subunits.
Discrete intermediate structures formed, cannot proceed unless previous structure formed.
- head, tail & tail fibres formed separately by sequential reactions, then assembled in ordered manner.
Concerted assembly (Polio in cytoplasmic compartment)
Structural subunits assemble productively hen nucleic acid present.
For Polio:
- +ssRNA translated immediately -> produces P1-3
- P1 has VP0, VP1 & VP3, spontaneously assemble into 5S subunit + polymerises forming 14S penton
- Pentamer stabilised by protein interactions + interactions mediated by mysterate chains on N-terminus of VP0
- Pentons can self-assemble, viral genome required to catalyse proteolysis of VP0 -> VP2 & VP4
-> produces infectious Polio from provirion
Concerted assembly of influenza A
HA, NA + M2 envelope proteins translated by ribosomes associated w/ ER
- delivered to plasma mem by host secretory pathway.
Ribonuclear proteins (nuclear export, M1) translated by free ribosomes, imported back to nucleus
- protein migrates to plasma mem to sites enriched w/ glycolipids, M1 protein genome binds C-terminal domain of HA
-> virus particle forms
8 genomic segments packaged into each capsid accurately
Self assembly of viruses (give examples)
TMV + Polio form spontaneously from capsid subunit + RNA
HA of influenza expressed in cells can bud spontaneously + form particles
Hepatitis B (HBV) surface antigen forms into virus like particles
What does assisted assembly require to be successful?
Need protein scaffolds to form capsid structure.
e.g. Herpes simplex, Adenovirus
- they establish intermediate structures, not present in mature virus but needed for accurate assembly of icosahedral virus
- subject to proteolytic degradation by viral proteases prior to entry of DNA genome into capsid
Assisted assembly in Herpes simplex
linear dsDNA encodes approx 80 proteins, membrane bound, icosahedral capsid.
Targets mucosal epithelial cells but can lie dormant in neurones. T=16, 969 subunits
pre-VP22a self association (forms internal scaffold stimulates VP5 (hexamers + pentamers) binding followed by triplet protein (VP23 + VP19C)
VP24 protease in core activated + cleaves short C-terminal sequence from scaffold protein
-> scaffold disintegrates + is further degraded which promotes conformational change allowing DNA entry
*only 1 UL6 portal protein - allows scaffold ejection + DNA entry into virus
List 3 requirements for ensuring successful infection
1) sufficient viral particles (shedding)
2) cells at primary infection site must be accessible, susceptible + permissive
3) local host of antiviral defences must be absent or initially defective
How do virions defend against hostile environments?
- large number of virions overcome sensitivity to heat, drying + sunlight
- many virions stable at low pH & protease resistant
- no exposure to environment via vector transmission or direct physical contact (e.g. zika, yellow fever)
Local vs systemic infection
Local - replication occurs at site of infection, no systemic spread
e.g. influenza, rhinovirus
Systemic - replication occurs at primary site of infection + disseminated via blood, lymph + nerves to secondary site of infection where replicate further. Can shed back into blood + disseminate further
e.g. measles, chickenpox
Viral entry via skin
Epidermis cannot support infection (dead keratinised cells)
Viral entry via skin abrasions, needle puncture, insect/animal bites
-> dermis + subdermal tissues highly vascularised
Viral entry via respiratory tract
e.g. rubella, influenza, mumps measles, varicella zoster
Alveoli targeted, entry into lymphatic/blood vessels. Goblet cells in mucociliary escalator has secretory IgA - aggregates pathogens.
Viruses either affect upper or lower tract
Viral entry via GI tract
Has innate defences - lysozyme in mouth, low pH + proteolytic enzymes in stomach
Systemic inc enterovirus, reovirus, adenovirus.
Localised infections inc coronavirus, rotavirus.
Polio virus binds M cells in Peyer’s patches to move across into lymphatics/blood, use it to cross epithelial mucosal barrier.
Enveloped viruses do not initiate GI tract infections except coronavirus (mainly naked)
Viral entry via urogenital tract
Less well protected, has mucus + low pH.
Minute abrasions in sex may allows entry via epithelial cells.
Systemic inc HIV, hepatitis B, herpes simplex can infect sensory + autonomic neurons.
Localised - human papillomavirus
Lymph provides access to blood stream -> haematogenous spread
Compare active and passive viremias
Viremia is when viruses enter blood stream + can access rest of body (either free or contained within infected cells - lymphocytes)
Active - replication in tissue prior to bloodstream access
Passive - viruses enter bloodstream without replication in host tissue e.g. direct inoculation by mosquitoes, don’t replicate until they reach permissive tissue
Compare primary and secondary viremias
Primary - release of virions after replication at initial site of entry into bloodstream, conc of virions low but allows spread to secondary sites
Secondary - replication at secondary sites results in large number of virions being released into blood -> spread to organs
List the different features of neural spread
Neurotropic - infect neural cells via neural/haematogenous route
Neuroinvasive - can enter CNS after infection of peripheral site (low in herpes, high in mumps + rabies)
Neurovirulent - can cause disease of nervous tissue -> neurological systems -> death (low in mumps, high in herpes + rabies)
How does neural spread take place and what are the two types?
Infection initiated in muscles or other innervated tissue. Can enter afferent /efferent nerve fibres + spread through axons to cell bodies + primary neurones.
- can only replicate in cell bodies
Anterograde spread: moves along MTs using kinesin (cell body -> axon)
Retrograde spread: moves along MTs using dynein (axon -> cell body)
e.g. Rabies targets muscle cells to initiate CNS infection + replicate
How do viruses cross the blood-brain barrier and what areas do they target for entry into brain?
e.g. Zika, Measles, West Nile, Polio
Can hijack lymphocytes or use transcytosis (vesicles), others replicate in endothelial cells lining blood vessel.
Meningeal blood vessel, cerebral blood vessels, choroid plexus blood vessel, meninges.
Poliomyelitis
Enterovirus, Picornaviridae (+ssRNA), 3 serotypes: Brunhilde, Lansing + Leon. Pseudo T=3 non-enveloped capsid, icosahedral symmetry.
- Humans only known reservoir, faecal/oral transmission (peaks warm months)
- Can enter CNS + replicate in motor neurones in spinal chord
Complications : post-Polio syndrome (25%-40%) 30-40 yr interval, caused by long term damage to motor neurones
Paralytic 1% cases (flaccid paralysis).
Bulbar poliomyelitis has max fatality as brain stem neurons involved.
Pathogenesis of Polio
Ingested + replicates in orapharynx + intestinal mucosal surface. Targets M cells w/ PVR CD155.
Enters via cervical/mesenteric lymph nodes -> viremia.
Can migrate to muscle (replicates) + reach motor endplate (access to CNS)
Moves down axon (12cm/day) - cytoplasmic C-terminal tail of CD155 associated w/ Tctex-1 (light chain subunit of dynein motor complex)
-> retrograde transmission
Replication:
-Polio cleaves translation initiation complex eIF4E, ribosomes cannot be recruited to capped mRNAs
- only uncapped Polio virus mRNA which has IRES is translated
Rabies virus structure
Neurotropic lyssavirus, -ssRNA genome encodes 5 proteins, membrane bound, helical.
Bullet shape - glycoproteins act as adhesin, animal to human transmission. Can be transmitted via latrogenic cornea transplant or saliva (bites)
Spreads by retrograde axonal transport.
-> zoonotic virus
Rabies pathogenesis & symptoms
Varied incubation 7 days - many years (depends of wound, inoculum size + distance from CNS)
Replicates in striated/connective tissue + enters peripheral nerves via NMJs (retrograde).
Can replicate in dorsal root ganglion + travels up spinal chord to brain.
Spreads to CNS in endoneurium of Schwann cells (anterograde transport)
Symptoms:
Furious form 80% infections.
Non-specific prodrome period: fever, malaise, anorexia, nausea, sore throat, myalgia + headache
- acute encephalitic phase: hydrophobia + excitement, virus sheds in saliva
- numb form (20%): weakness, flaccid paralysis
After onset, survival rarely > 7 days
How does rabies virus navigate muscle cells, NMJs and enter neurones?
Glycoprotein dimer binds nAchRs at post synaptic muscle membrane.
Entry via endocytosis, fusion w/ endosomal membrane, ribonucleocapsid released into cytoplasm.
Virus moves across NMJ to neurons + enters via neural cell adhesion molecule (NCAM)
Either via capsid release or whole virus remains in endosome + transported to cell body.
Retrograde axonal transport to cell body
What characterizes persistent smoldering viruses?
They replicate constantly
E.g. lymphocytic choriomeningitis virus
What are acute infections?
Short duration infections that are cleared in 1-2 days. Eliminated by immune system.
Transient then adaptive immune response (long lasting)
E.g. rhinovirus, rotavirus + influenza, rabies, polio, measles
What are latent viruses?
Viruses that can exist in a non-replicative form and are activated by environmental factors
E.g. Herpes simplex
What is the role of innate defenses in viral infections?
They help eliminate virions and infected cells early on
E.g. interferon gamma by cytokine response
What are examples of zoonotic infections?
- COVID
- H5N1
- Ebola
- Zika
What is the duration of persistent infections?
They can last a long time, often characterized by slow progression
Fill in the blank: The threshold level of _______ is required to activate the adaptive immune response.
[virus]
What is the difference between innate and adaptive immune responses?
Innate is immediate and non-specific, while adaptive takes time and is specific
Short vs long incubation
Short - virus replicates + causes disease at primary site of infection
e.g. influenza (1-2 days)
Long - requires sytemic spread of virus from primary site (primary + secondary viremia) e.g. measles, chckenpox
Descirbe an inapparent acute infection
Succesful acute infections that have no/mild symptoms.
- enough to maintain infection but insufficient to cause disease
- caused by well adapted pathogen
- detcted by increase in antiviral antibodies
> 90% Polio infections inapparent
Why is Tamiflue useless unless given early on?
Most acute infections complete before immune response detected.
- virus has spread to next host
- difficult to diagnose prior to symptoms
Tamiflu beneficial within 2 days
Define R & r
r is the growth rate in a population
R is the number of people 1 person will infect
Measles structure + characteristics
-ssRNA human specific, evolved from rinderpest, only 1 serotype
R = 16 so very contagious - 40 mil infections a yr
large pop required to maintain the virus
Pathogenesis of measles
Infects epithelium, infected alveolar macrophages + dendritic cells migrate to lymph nodes where B + T cells infected (1 viremia)
Infected B/T cells replicate in spleen, thymus + lymphoid organs then re-eenter circulation (2 viremia)
Can spread to all body surfaces - respriratory (cough), mouth (Kopliks spots, rash)
Timeline of measles infection
Most infectious 2-4 days before & 2-5 days after rash develops
- viral numbers largest in prodromal phase (non-specific symptoms), when virus shedding lots
- 1o & 2o viremia effectively incubation period
Outcomes & treatment of measles
- affected by noruishment e.g. vitA can reduce mortality
- immunosuppression can cause secondary infections (60% deaths due to pneumonia)
- subacute sclerosing panencephalitis (SSPE), degeneration of NS - rare
- immune amnesia (memory cells destroyed, 2-3 yrs)
100 a year chronically disabled (chronic encephalitis)
- endmeic transmission stopped in 2000 by MMR vaccine
Wakefield 1998 -> decreased immunisation, so increased outbreaks
Why do acute infections reoccur?
- antigenic variation by mutations of structural proteins (antiody resistance)
- strcutural plasticity, can tolerate many a.a.cid subs + remain infectious (rhinovirus, 100 serotypes)
WHEREAS polio, measles + yellow virus have rigid structure + few serotypes so long asting effective vaccines used
Antigenic drift
Error prone replication of RNA genomes in viruses (no proof reading)
Cna improve/reduce fitness
Antigenic shift
Major change in surface protein of a virion following acquisition of new genes.
e.g Influenza A - new pandemic strain
influenza - genome reassortment when mutliple viruses coinfect cells
porcine cells can be infected w/ bird + human influenza
List the initial symptoms of influenza.
Incubation 1-5 days
Abrupt onset headache
* Chills
* Dry cough
* High fever
* Myalgia
* Malaise
* Anorexia
When does fever typically decline in influenza?
Day 2-3, gone by day 6
What antiviral drug is effective against influenza if taken within 2 days of infection?
Tamiflu
What are some complications associated with influenza?
- Primary viral pneumonia
- Secondary bacterial pneumonia
- Generalized muscle pain
- Cardiac involvement
- Reye syndrome
What is Reye syndrome associated with?
Encephalopathy and liver problems
What role do cytotoxic lymphocytes play during an influenza infection?
They cause damage while clearing the infection
What is haemagglutinin (HA) in the context of influenza?
Trimeric glycoproteins that promote adhesion of virus to alpha 2,6-linked sialic receptors
What is the function of neuraminidase (NA) in influenza?
Catalyzes hydrolysis of terminal sialic residues from newly formed virions and host cell receptors
What are the subtypes of influenza A based on HA and NA?
18 HA subtypes and 11 NA subtypes
Name the three combinations of influenza A that circulate in humans.
- H1N1
- H2N2
- H3N2
What is antigenic drift in the context of H1N1?
Variants appear every 3-8 years
How often do variants of H3N2 appear?
Every 2-5 years
How many serotypes of influenza B exist?
1 serotype
2 major lineages are Victoria and Yamagata - diverged 1970s, Yamagata outcompeted by COVID
What is the mutation rate of influenza B compared to influenza A?
2-3x slower
What drives zoonotic transfer?
Mutations - some may have gene that allows progenitor jump into new host
- relies on standing variation in original host
de novo mutations in new host promote adapation -> undetected sustained transmission
What are the two classes of HIV that cause clinically indistinguishable disease?
- HIV1
- HIV2
Both are members of the lentivurus class of retroviruses
HIV1 is responsible for the global pandemic, while HIV2 is restricted to West Africa.
What is the origin of HIV1 and HIV2?
- HIV1: SIV from chimps/gorillas
- HIV2: SIV from Sooty Mangabeys
Both viruses integrate into the host genome as a provirus.
What significant health issues were reported in young males in San Francisco and New York City in 1981?
- Kaposi’s sarcoma
- Pneumocystis pneumonia
Luc Montangnier isolated HIV 1984
These conditions were indicators of AIDS.
What type of cells does HIV destroy, weakening the immune system?
CD4 T-cells
The depletion of these cells leads to immunodeficiency.
How many people were living with AIDS in 2023?
39.9 million
This is an increase from 29.4 million in 2001, attributed to higher survival rates.
What is the structure of the HIV genome?
2 identical copies of a 9749 nucleotide (+) ssRNA molecule, encoding 15 proteins
The genome is key to the virus’s replication and function.
What is the significance of the gp120 spikes in HIV?
Enables targeting of CD4
These spikes are crucial for the virus to attach to host cells.
What protein forms the capsid structure of HIV?
p24 protein
This protein is key for diagnosis of HIV.
What is the composition of the HIV capsid?
- 216 protein hexons
- 12 protein pentons
The pentons are formed at positions where tightly rounded corners occur on the capsid.
What role does water play in the HIV capsid?
Associates w/ capsid - helps the virus dissociate when inside the cell
This is important for the viral entry into host cells.
Genomic organisation of HIV-1
9 ORFs but 15 proteins made -> cleavage of 3 primary products (Gag, Pol + env polyproteins)
Separated ORFs subject to alternative splicing -> splice variance allows tat & rev to associate w/ each other
Function of Gag, Pol & Env in HIV-1
Gag - matrix, capsid nucleocapsid, p6
Pol: reverse transcriptase (RNA -> dsDNA), integrase, protease (cleaves Pol + Gag encoded polyproteins)
env - Gp160 proteolytically cleaved -> gp120 + gp41by a furin (golgi-dependent protease)
TAT protein
Trans-activator trancription
Binds downstream sequence of LTR + stimulates transcription, secreted + taken up by healthy cells -> upregulates CCR5 + CCrX4
REV protein
Regulator of Virion protein expression
Dictates whether RNA translated or packaged into virions.
High - protein synthesis rises
Low - ensures full length RNA for packaging
NEF protein
Negative regulatory factor
- myristolated, anchored to inner mem surface
- binds C-terminus CD4, endocytosed + destroyed preventing super infection cells
- prevents MHC class I presentation by binding golgi mem (undetected by host immune cells)
- increases NFkB activation, increasing HIV transcription
- triggers apoptosis bystander T cells
VIF
Protein infectivity factor- protects HIV from antiviral defences
ApoBec3G polyubiquinylated -> degraded in proteasomes
IF VIF absent, ApoBec3G binds viral RNA + converts C->U in synthesised cDNA -> hypermutation (C to A transversion) or degradtaion of cDNA
It would also inhibit reverse transcription
VPU protein
Viral protein U
- enables release of HIV from plasma mem
- prevents export of DC4 to cell surface, trasp it in ER for degradation
VPR
Viral protein R
- promotes entry of cDNA into nucleus -> cell cycle arrest
Describe the phases of HIV infection
Acute - virus tire rises + initial fall in CD4/rise in CD8 T cells, M tropic (CCR5) viruses predominate (homogenous pop)
Asymptomatic phase - 3-4 months after infection, replication continues in lymph nodes, virus binds follicular dendritic cells, latent reservoir in quiscent T cells, accumulated mutations -> population becomes more heterogenous
Development of AIDS - CD4 count below 200/mm3 blood, virus titre rises again, immune competecy declines -> opportunistic infections.
Attachent + entry of HIV
- specifc binding gp120 to CD4+ -> confromational change exposes variable loop 3 promoting co-receptor binding
- gp120 dissociates from gp41 which assumes haiprin conformation
- allows membranes to fuse (fusion target of antiviral drugs -> T-20/enfuvirtide)
Env protein is only target of neutralising antibodies -> drives evolution in response to selection pressure
Reverse transcription in HIV
Capsid enters cytoplasm + reverse transcription occurs in capsid.
Pro-virus (DNA) imported + integrated in nucleus. Cell does not need to be actively replicating as VPR as nuclear localisation signal in C terminus -> binds nuclear import receptors.
HIV matrix + integrase also have NLS
Integration in HIV
Virus uncoats at nuclear membrane + DNA enters nucleus (both linear + circular forms found).
Linear viral genome cropped by integrase (2bp removed) revealing 5’-TG & 3’-CA
- dsDNA then integrates into chromosome using integrase
Transcribed using host Pol-II
Assembly + maturation of HIV
Gag proteins asscoiate w/ plasma mem + RNA genome.
- core assembled
- fusion of membrane releases immature non-infectious viral particle
Proteolytic cleavage of Gag & Gag-Pol polyproteins completes maturation -> infectious
SO protease key target for antivrial drugs (Atazanavir ATV, Darunavir DRV)
Why can HIV infection not be mimicked in macaques?
Trim5a blocks HIV replication by binding to capsid + targeting it for degradation by proteosome.
HIV induced apoptosis
Infected activated T cells transcribes caspase-3 triggering cell death.
5% cell death
HIV induced pyroptosis
Infected quiescent T cells dont have enough nucletoides for reverse trasncription.
- incomplete rev trascripts detected by IFI16 binding - activating caspase-1
- caspase-1 cleaves pore forming gasdermin -> osmotic lysis
- pro-inflammatory cytokine (IL-1B) attracts more T cells -> exacerbates process
What causes clinical latency?
Strong immune repsonse to acute phase -> decline of virus in blood.
BUT still replicates in lymph nodes dendritic cells + macrophages.
Difefrent latent reservoir in quiescent memory T cells (no replication)
What is the set point?
Stable level of HIV in the plasma of an infected person after the initial sharp increase in viral load during acute phase
-> determines speed of progression (length of asymptomatic period)
Rotavirus general structure
11 segment dsRNA genome
VP7- G antigen, VP4 - P antigen
10 G + 11 P serotypes - 42 P&G combinations identified
Causes 30-50% diarrhoea in infants worldwide
Pathogenesis of rotaviruses
Transmitted by faecal/oral route through fomites, very stable on environemntal surfaces.
Resistant to hand washing -> 3 layer capsid structure
Infants at risk of dehydration - electrolyte replacement therapy essential
Incubation period - 1-3 days, infection can last 5-7 days
Role of M1 protein in inluenza
tethers the ribonucleoprotein to HA and NA at the plasma membrane
terminates transcription and translation of the viral genome
controls budding of the mature virus from the cell
Latent/proviral infection characteristics
No replication, some transcripts made -> only proteins for maintaining latency + inactive state
- initial acute infection followed by quiescent phase
- reactivation can occur + always sheds virus but symptoms not always present
Persistent/chronic infection characteristics
Long term infections w/ low level virus production
- not cleared effectively by adaptive immune response
LCV - Lymphocytic Choriomeningitis virus
e.g. LCV mice infected congenitally or at birth -> shed virions, not recognised as foreign + infection not cytopathic
What causes persistance?
- Inhibition of apoptosis
- Neutralisation of CTL response prevents detection
- Inhibition of T cell activation by modulation of MHC class I & II antigen presenting pathway
- Ineffective stimulation of host iterferon response
- modulation of viral gene expression
Give some examples of latent viral infections
(gamma) Epstein Barr - novel transcription + replication pattern, no new viurs but genome replicates
(B) Cytomegalovirus - restricted transcription, genome does not replicate
(a) Herpes Simplex & VZV - restricted transcription, genome does not replicate
^All Herpesviridae
Describe the structure of alphaherpes
200nm diameter, linear dsDNA genome, icosahedral nucleocapsid.
Lipid envelope w/ 10 viral glycoproteins, tegument layer - 15 viral proteins
Very well adapated to humans
PNS is a latent reservoir - neurones do not replicate DNA or divide (immunologically priveliged niche)
HSV-1 pathogenesis
- spreads locally in epithelial cells, taken up by lymphatic system.
- replicates in oral mucosal ep. cells + enters nerve termini of sensory neuronewhich innervate primary infection site
- HSV travels to neuron cell bodies in trigeminal ganglia, via fast axonal transport
- lytic genes repressed + latency occurs
How does HSV-1 induce latency?
In sensory ganglia, it become slatent as a non-integrated, nucleosome associated episome in host cell nucleus.
Transcription of virla genome silenced apart from LAT
- LATs prevent expression of genes needed for lytic phase of HSV-1 replication
LAT - latency associated transcript
What is the role of gD in HSV1 infection?
gD interacts with nectin-1
gD is a glycoprotein that facilitates the binding of the virus to host cell receptors.
What extracellular matrix components does HSV1 bind to?
gB and gC bind heparin sulphate and chondroitin sulphate
These components are crucial for the initial attachment of the virus to the host cell.
Which HSV1 glycoproteins mediate membrane fusion?
gD, gB, gH, and gL
Nucleocapsid and tegunent proteins then released into cytoplas
These glycoproteins play a key role in the fusion of the viral and plasma membranes.
How are HSV1 nucleocapsids transported to the nucleus?
They attach to microtubules along with VP16 (teg protein)
This transport mechanism is vital for delivering the viral genome to the nucleus.
What does VP16 stimulate?
Transcription of immediate early genes
Products translated and imported into nucleus - activate transcription of proteins need for DNA replication
VP16 is a tegument protein that activates early viral gene expression.
What are long concatomers of DNA in HSV1 replication?
Made in replication, encode structural and assembly proteins
These serve as templates for late gene expression.
Where are mature glycoproteins transported?
To the Golgi and then to the plasma membrane
This process is important for the final assembly and release of the virus.
Fill in the blank: The viral genome is delivered to the nucleus, activating viral gene expression dependent on _______.
Tegument protein VP16
VP16 is crucial for initiating the expression of early genes.
Latency of HSV-1 in neurones
Viral genome circularise + transcription repressed (except LAT), ICP0 repressed + VP16 does not enter nucleus
LAT transcript spliced -> lariot structure, trasncripts produce ant-sense RNAs (4 miRNAs + 2 sRNAs) that prevent ICP0 transcription
- 2 of miRNAs prevent translation of 2 proteins needed for apoptosis so cell not killed
vhs (virion host shutoff) encoded by HSV gene UL41 is mRNA specific RNAse - rapidly shuts down host cell protein synthesis
no viral proteins produced so not detected
Why does HSV-1 normally remain in the latent phase?
tegument proteins transported ineffficiently (interaction w/ fast axonal transport system - genes not upregulated so no VP16 to exit latent phase
Reactivation poorly understood - cortisol maybe involved (stress + chromatin remodelling)
-> requires de novo VP16 synthesis, may be done by relapse of chromatin repression elements
VP16 not transported effiiently to nucleus (trasncriptional silencing)
Varicella Zoster & latency
125kb genome encode 68 ORFs
Primary infection -> chicken pox, then remains dormant in trigeminal + dorsal root ganglia
Can reactivate as shingles later in life (10-20%)
Viral gene transcription epigenetically regulated + restricted to ab 5 genes.
- ORF63 inhibits apoptosis + most abundantly expressed
- accumulates in neurone cytoplasm
Epstein Barr virus & latency
172kb linear dsDNA, subclinical in children - causes infectious monnucleosis in 50% adolescents. 80% adults seropositive
- lies latent in non-proliferating B-lymphocytes
- subpahryngeal epithelia cells infected by saliva
EBV genome maintained as circular episome + associates w/ ncuelosomes. It is methylated at CpG residues
- expresses only LMP2A & EBNA-1
- infected B cells sequestered to bone marrow + lymphoid tissues
Not seen by CTLs or antibodies - virus only procuded in small fraction of cells
Reactivation involves Zta protein - activates host gene erg1
What are the stages of viral infection relevant for antiviral intervention?
1) Binding to viral receptor
2) Penetration of cell
3) mRNA function
4) DNA/RNA synthesis
5) Viral assembly
6) Transport & release of virus
What is needed to identify an agent that can target attachment and entry of a virus?
Identification of agents that mimic viral adhesin or receptor to bind to adhesin.
What is Maraviroc and its function?
A CCR5 antagonist that prevents interaction with gp120.
What does Enfuvirtide bind to and what is its effect?
Binds gp41 and interferes with HIV’s ability to catalyze membrane fusion.
What is the role of Amantadine in antiviral treatment?
Interferes with formation of pores in influenza virus particle by M2 protein.
Targets uncoating
True or False: Widespread resistance in influenza A and B is due to single amino acid mutations in M2.
True
What is Pleconaril used for?
An anti-rhinovirus drug that binds hydrophobic pocket in VP1 to prevent uncoating.
What is Acyclovir and its mechanism of action?
A pro-drug activated by phosphorylation, treating Herpesviridae by causing chain termination in viral DNA.
What type of analogue is Acyclovir?
Guanosine analogue.
What is the function of Zidovudine (AZI)?
An antiretroviral drug that terminates DNA synthesis to abort replication.
T analogue, highly toxic + y-Pol in Mt DNA highly sensitive
What type of analogue is Zidovudine?
Thymidine analogue.
What is Molnupiravir and its mechanism?
A pro-drug metabolized into nucleoside analogue N4-hydroxycytidine 5-triphosphate, incorporated into viral RNA instead of cytosine.
What does Molnupiravir mimic in viral RNA?
Cytosine, but can swap between cytosine and uracil-like structures.
What is the function of protease inhibitors in antiviral therapy?
Competitively inhibit viral protease, preventing maturation of released virions.
HIV - atazanavir + darunavir
SARS-Cov2 - ritonavir
Name a protease inhibitor that targets SARS-CoV-2 protease.
Lopinavir/ritonavir.
What are integrase inhibitors used for?
To block further spread of HIV virus (raltegravir) and used in salvage therapy when resistance to other drugs has occurred.
Must be combined with other drugs that target specific steps.
How many integrase inhibitors are currently licensed?
4 drugs licensed + 1 in development with a greater half-life.
The drug in development is intended for pre-exposure prophylaxis.
What did the discovery in 1974 reveal about DANA?
DANA was discovered to be a neuraminidase inhibitor.
This discovery led to the development of antiviral drugs targeting neuraminidase.
What are Relenza and Tamiflu?
DANA derivatives with high specificity for the catalytic site of neuraminidase, inhibiting the release of budding viruses from cells.
Relenza is delivered by inhalation, while Tamiflu is taken orally.
Against which viruses are Relenza and Tamiflu effective?
Effective against influenza A & B.
Both drugs target the neuraminidase enzyme critical for viral replication.
What is Baloxavir marboxil?
A new anti-influenza drug delivered orally that acts as a cap-dependent endonuclease inhibitor.
It stops viral replication within 24 hours, faster than Tamiflu.
What does the endonuclease do in viral mRNA synthesis?
Snips off the 5-end of a host capped, methylated mRNA which serves as a primer for viral mRNA synthesis.
What is the ‘Kick & Kill’ strategy in HIV treatment?
Triggers transcription of latent HIV virus, allowing latently infected cells to be targeted and killed by the immune system.
SAHA (Suberoylanilide hydroxamic acid) is one drug that facilitates this process.
- inhibits HDAC so acetylated histones accumulate
- induces apoptosis by cleaving Bid protein + produces reactive oxygen species
What is BCX4430?
A synthetic adenine analogue that inhibits replication of 20 viruses including Coronavirus and Ebola.
It is considered a broad-spectrum antiviral.
What is T-705 known for?
It is effective in vitro and in vivo with ongoing human trials for treatment of Influenza and Ebola.
T-705 is another broad-spectrum antiviral.
What is CMXO01?
A lipid conjugate of the nucleoside analogue, cidofovir, showing activity against multiple DNA viruses including Herpesviridae and Papillomaviruses.
What do drugs targeting cytokine storms aim to treat?
They aim to treat general symptoms that accompany acute viral infections.
Cytokine storms can lead to severe inflammation and other complications during viral infections.
Immunosuppressant - azathriopine
How are antiviral drugs discovered?
Mechanism based cellular screens targeted to a particular viral encoded enzyme or interaction.
e.g. transcription, membrane fusion
High throughput screens allow large numbers of compounds to be automatically screened - uses fluorescence markers.
Principle of live attenuated vaccines & potential problems
From attenuated straisn devoid of pathogenicity but can induce immune response.
- forced to undergo mutations in unnatural host (monkeys) or at sub optimal temps
Problems: under-attenuation, revrsion, instability, heat labile, contamination of viral cell culture.
measles vaccine, forced human specific virus to replicate in chick embryo lines under 32C
Attenuated influenza vaccine
Cold adaptation in co-infected chicken kidney cells at 25C (master strain) -produced in embryonated eggs.
- only healthy individuals 2-49yrs old
- administered by nasal spray
- cross reactive againsts drifted strains
- IgG + IgA production, strong immunity
Attenutaed polio vaccine
Sabin
Type 1 & 3 generated by serial passage in mokey cells for reduced neurovirulence strains
Type 2 is naturally ocurring attenuated isolate, requires 3 doses -> >98% immunity for recipients
BUT oral polio vaccine can revert to nurovirulent form + shed in faeces, 1/750,000 associated w/ paralytic polio
used where risk of wild Polio very high, replaced by IPV as incidence decreased
Priciple of inactivated vaccines
Inactivated by heat or chemical (formulin, B-propiolactone)
Need optimal treatment for sufficient immunogenicity.
- excessive treatment can destroy immunogenicity
- insufficient can leave virus still pathogenic
requires boosters as less immunogenic
Inactivated influenza virus
Trivalent vaccines made in embryonated eggs BUT takes several weeks, viral yield can be poor + major egg supply limitations.
Safe + can be given to anyone, requires adjuvant (alum used in 5 influenza vaccines) to boost immune response.
Inactivated Polio vaccine
1955 Salk injected inactivated Polio
Based on 3 wild virulent reference strains, grow in monkey kidney cells (Vero cell line is inactivated w/ formulin)
IgG mediated immunity in blood prevents viremia, does not affect motor neurone
preferred in regions w/ no wild polio risk
Inactivated vaccines differences w/ live attenuated
- needs higher + multiple doses
- needs adjuvant (e.g. alum)
- shorter immunity duration (unknown)
- no IgA response
- poor cell mediated immune response
- reversion to virulence not possible
- possible for incomplete inactivation
live vaccine -> replication of virus in host
Principles of subunit vaccines
Purified immunogenic viral surface proteins used to iinduce immunity via antibody interaction.
1st HepB vaccine used HBsAg purified from blood of carriers - has excess coat proteins -> form speherical + tubular particles
- cloned subunit produced in yeast cells
extensive purification needed to remove infectious viral particles
Pro and cons of subunit vaccines
Pros:
- suitable for those w/ comproised immune systems
- no live components so does not cause disease
- relatively stable
Cons:
- complex to manufacture
- needs adjuvants + boosters
- determining best antugen combination takes time
Human Papillomavirus (HPV) subunit vaccine
Capsid has 2 VPs: L1 (major) + L2 (minor)
Recombinant HPV L1 used to assemble hollow virus like particle. Cloned into baculovirus vectors + expressed in insect cells
- NHS uses Gardasil (for 9 serotypes)
- L1 expressed in yeast/baculovirus infected insect cells
- L1 folds correctly + self assembles into VLPs when expressed in eukaryotes
HPV16 & HPV18 cause 70% all cervical cancers
FluBok subunit vaccine
Quadravalent rHA vaccine (2 type A & 2 type B)
Produced using baculovirus exoression system.
- produced 3x more HA than trivalent inactivated vaccine
- no egg protein or preservatives (mercury), so higher doses w/o risk of side effects
Approved for seasonal flu 2020/21 in UK
Principles of mRNA vaccines
Synthetic mRNA directs production of immunogenic protein.
Can include viral replicase genes -> amplify intracellular RNA
mRNA contains modified nucleosides:
uridine replaced by pseudouridine, C replaced w/ 5-methyl cytosine
- prveents RNA sensors (TLR7/8) recognising foreign RNA + triggering inflam response
Incorporation in lipid particles prevents degradation + helps endocytosis into APCs.
Describe the 3 main COVID-19 vaccines
Pfizer - nucleoside modified RNA encoding spike protein in lipid nanoparticles (stored at -70C)
Moderna - modRNA encoding spike protein in lipid nanorparticle, stored at 2-8C, can be frozen + stable for several hours at room temp
AstraZeneca - replication deficient chimp andenovirus vector carrying full length spike protein gene (DNA vaccine)
mRNA based universal influenza vaccine
Ideally gives cross strain protection
- periodic vaccination needed that targets common epitopes
vaccine using conserved stalk portion of HA started clinical trial in US may 2023 so bypasses serotype variation
-> gives immune response to 20 diferent strains in mice + ferrets
Herd Immunity
Virus stops spread when probability of infection drops < critical threshold
e.g. measles is 93-95% (very infectious)
Can be reduced by social opposition
e.g. 2003 Nigerian religious leaders claimed polio vaccine had contraceptives derailing vaccination campaign so incidence rose
e.g. MMR & autism Wakefiled 1998
no vaccine 100% effective
