What are the 3 domains
- Archaea
- Eucarya
- Bacteria
Different types of eucarya
- Fungi
- Plants
- Animals
- Protists
What are the key reservoirs of biomass and nutrients for all life?
Bacteria and archaea
Why are there so many microorganisms
- Rapid growth rate
- Many chances of speciation through random mutation
- Exchange of genetic material
- Every niche is occupied
What is a phototroph
Gets energy from light
What is a chemotroph
Gets energy from chemicals
What is an organotroph
Uses organic compounds as electron donors
What is a lithotroph
Uses inorganic compounds as electron donors
What is an autotroph
Uses Co2 as a carbon source
What is a hetrotroph
Uses oranic carbon as a carbon source
Two types of primary nutrients
Macronutrients (carbon, nitrogen) and micronutrients (trace metals)
4 different stages of a growth curve
- lag phase (adaptions to new conditions)
- exponential phase
- stationary phase (Limitations in nutrients and a build up of waste products)
- death phase
Different measures of growth in microorganisms
- Cell number (haemocytometry, dilution plating)
- Optical density (turbidmetry)
- Fresh/dry weight
- Protein
- DNA
How to identify microorganisms
- Staining and microscopy
- Growth on selective media/ differential media
- Testing enzyme activity
- Characterising of cell constituents (lipid, cell wall, components)
Modern identification is based on sequencing of conserved genes.
How does selective media identify microorganisms
Allows the growth of some microorganisms
How does differential media identify microorganisms
Based on growth and appearance on that media
Testing enzyme activities towards pathogens
- Culture the organism
- Cell is re-suspended in a buffer
- Test wells are inoculated
- Test strip is used to see enzyme activity by colour change, which is compared to a database
- Used to distinguish between pathogenic strains and benign strains
Surface origin hypothesis
- On the surface there was a primordial soup, organic compounds formed by chemical reactions and electrical activity caused by meteor strikes
- Organic acids came together to form amino acids and nucleotides
- Theory not likely due to hostile conditions on the surface
Subsurface origin
- Life begins in hydro thermal vents where compounds mixed at high temperatures
- Self replicating RNA forms and enzymatic proteins
- DNA forms and leads to the evolution of biochemial pathways
- Divergence of lipid biosynthesis, cell walls and cell type
- Formation of early bacteria and early archaea
What are the landmarks in biological evolution
- Early life is dependent on H₂ and Co₂
- Energy and carbon metabolism diversifying
- Phototrophy using H₂S as am electron donor
- Evolved into oxygenic photosystem using H₂O
Define phylogenics
How related organisms are to each other
Molecular sequences used in phylogenics must be
- Universal
- Contains conserved and variable regions
- Not subject to gene transfer
- Must be truly homogeneous (perform the same function)
Tree of life use comparative ribosomal RNA
Basic evolution of eukaryotes and the 2 theories
- Mitochondrion appeared in a proto eukaryotic cell
- Nucleus formed
- Plants formed when chloroplasts came about
Endosymbiont theory and hydrogen hypothesis
What is the endosymbiont theory
- Mitochondria was formed due to the incorporation of aeorbic chemoorganotrophic bacteria into a host cell
- Chloroplasts formed due to the incorporation of phototropic cyanobacteria into a eukaryotic cell
What is the hydrogen hypothesis
Symbiotic association of an archaeal host using H2 as energy source with an aerobic bacterium that produced hydrogen as a ‘waste’ product.
Higher organism species definition
They interbreed to produce fertile offspring
Why is it hard to classify bacteria species
- Asexual reproduction
- Lateral gene transfer
- Phenotypic and genotypic plasticity of microorganisms
What is polyphasic bacterial taxonomy
Taxonomy taking in account:
- Phenotypic analysis (Morpholical, metabolic, physiological characteristics)
- Genotypic
- Phylogenetic (evolutionary links)
Gram staining procedure
- Spread culture thin and air dry
- Add crystal violet
- Fix with iodine
- Wash with alcohol
- Counter stain with safrarin
- Positive appears purple, negative appears blue
What does a barophile require
a high pressure
What does a halophile require
very salty conditions
What is a microaerophile require
requires low oxygen concentration
What does a psychrophile require
low tempertures (<15 degrees)
What does a mesophile require
“normal” temperature (15-45 degrees)
What does a thermophile require
high temperature (>50 degrees)
What does a hyperthermophile require
very high temperature (>80 degrees)
Name some colony based characteristics
Colony shape, margin, elevation, opacity, texture, pigmentation, odour
What is molecular analysis FAME (fatty acid methyl ester)
It determines fatty acid profile of membrane lipids by:
- Grow under standard conditions
- Extract lipids and chemically modify to methyl esters
- Analyse products by gas chromatography
Drawbacks of FAME
- Fatty acid profile depends on growth conditions which need to be standardised
- Not all strains can be cultured in these conditions
4 different types of genotyping analysis
- DNA-DNA hybridisation
- DNA profiling
- Multilocus sequence typing (MLST)
- GC base ratios
What is DNA-DNA hybridisation
Genome wide comparison of sequence similarity
What is DNA profiling
Producing DNA fragment patterns for comparative analysis
What is multilocus sequence typing
Sequencing several housekeeping genes and assign different alleles to different strains
What are the outer layers of a gram positive cell (outside to inside)
- Capsule, s-layer
- Cell wall (peptidoglycan)
- Periplasmic space
- Cytoplasmic membrane
What are the outer layers of a gram negative cell (outside to inside)
- Capsule, s-layer
- Outer membrane
- Periplasmic space with a layer of peptidoglycan
- Cytoplasmic membrane
What is a capsule and what are its roles
- It is a polysaccharide layer outside of the cell wall
- Prevents cell dehydration
- Involved in the attachment to surfaces
- Involved in the capture of nutrients
- Acts as carbon store
What is the s-layer
- A layer of protein and glycoprotein external to the cell wall
- Protects against ion and pH fluctuations as well as osmotic stress
What is peptidoglycan (PG) made of
- Alternating residues of NAG and NAM which are cross linked by amino acid side chains
- Has D-amino acids which protects against degradation by proteases
- It retains the stain in gram ve+ cells
What is a lysozyme and what is its significance
- An antibacterial enzyme
- Degrades 1-4 glycosidic bonds in pepidoglycan back bone
- Loss of PG makes the cell sensitive to changes in osmotic changes
- Important om host defence against bacteria
What are teichoic acids
- Only found on gram positive cells
- Ribitol or glycerol polymers joined by phosphate groups
- Covalently bound to peptidoglycan
- Gives the cell wall a negative charge
- May help the cell to obtain Mg2+ and Ca2+
roles of sterols and hopanoids
- Hopanoids in bacteria, sterols in eukaryotes
- They are rigid molecules that stabilise the membrane structure
The 2 ways the outer-membrane is linked
- Braun’s lipoportein
lipoproteins covalently linked to peptidoglycan and embedded in the outer membrane - Adhesion sites
Where inner and outer-membrane adheres
What are archaeal cell walls
- Typically has no outer membrane
- No peptidoglycan
- Instead has a polysaccharide called pseudomurein which is similar to peptidoglycan
- If the archaeal cell doesn’t have pseudomurein it will contain other polysaccharides
What is the lipopolysaccharide (LPS) composed of?
- Lipid A
- Core polysaccharide
- O side chain
Also called an endotoxin when it is free in the host
Significance of the O-antigen
- Variable region responsible for antigenic make up of bacteria
- Different O serotypes link to different diseases
- Responsible for species specific attachment
- Smooth and rough variants
Function of the different components in the LPS
- Lipid A stabilises the outmembrane
- Core polysaccharide is negatively charged, reducing permeability of hydrophobic substances
- Protects against host defences
- O antigen is used as a key diagnostic tool due to its variability
Function of endotoxin and when is it produced
- Produced by pathogens during cell division or by lysis of bacteria
- It primes the immune system against a pathogen (Immunogenic)
How are endotoxins tested for
By an LAL assay, where blood cells from Limulus polymephus clot when they come in contact with an endotoxin
Important properties of endotoxins
- Heat stable
- Toxic in nanogram amounts
- Triggers release of cytokines and activates transcription factors
- Results in inflammation, fever, vasodilation
What are porins
- Highly conserved transmembrane proteins that form water filled channels
- Made out of 3 identical subunits
- Most are nonspecific channels but some are specific
- Very stable due to a salt bridge
Function of the periplasm
- Nutrient acquisition
- Energy conservation, electron transport proteins
- Peptidoglycan synthesis
- Binding proteins that deliver compounds to ABC transporters
2 pathways that transport protein into the periplasm
Sec pathway and TAT pathway
What is the sec pathway
- Exports nascent polypeptide through cytoplasmic membrane using a translocase
- Folding of protein occurs after translocation
What is the TAT pathway
-Exports fully folded enzymes across the cytoplasmic membrane
What is a flagella
Long thin extracecllular helical structures that aid in motility. Proton motive force causes conformational changes in the motor.
Structure of the flagella
- Cap
- Filament (made out of flagellin)
- Has a base known as the hook
- Ring structure (transfer of protons through the ring structure drives the motor)
rings and hooks are attached to the membrane
Synthesis of flagella
- MS and C rings form
- Motor proteins form
- P and L rings form, the hook and cap
- Filament grows through the flow of flagellin through the hook
What are the different types of flagella
- Single flagella
- Flagella at opposite poles
- Multiple flagella at opposite poles
- Have flagella anywhere on the cell
What are the different motility patterns
Run and tumble. It is a fairly random process, but bacteria dont move in random directions.
What can motile molecules move towards
- Oxygen
- Nutrients, away from toxins
- Move along magnetism line
- Light
Role of methylated MCP
It is “bacterial memory” and remember previous concentrations of attractant and repellent
- High conc. attractant: Methylated MCP results in short runs and tumbling to stay in a “good” environment
- High conc. repellant:Methylated MCP results in longer runs and less tumbling to leave “bad” environment
What is gliding motility
Bacteria glides across a surface by the lateral movement of outermembrane protein. Does not need flagella or pili
What is twitch motility
Type 4 pili extend from the surface than retracts itself, pulling itself along. Powered by ATP hydrolysis. Pili is different to flagella.
What are gas vesicles
- Protein vesicles containing gas, which confers buoyancy to the cell
- Found in planktonic bacteria and some archaea
- Cells can float towards light or oxygenated water
What are fimbriae/pili
- Surface appendages involved in bacteria adhesion to surfaces
- Allows pathogens to attach to tissues
- Aid resistance to phagocytosis
- Are antigenic
Type 1 and type 4 pili
- Type 1: Thinner and shorter than flagella, hundreds to thousands per cell
- Type 4: Had an adhesive tip that binds to a glycolipid or glycoprotein. Pili pulls bacerium closer to the host cell.
Structure of type 1 pili
- FimH is the tip adhesin
- FimF and FimG link FimH to FimA
- FimA is the fimbriae
- FimC is a chaperone
- FimD is an usher protein (catalyses FimA polymerisation at the base)
What is the F pilus
- Pilus connecting two bacteria cells
- Involved in the transfer of genetic material, transfers DNA in the form of a plasmid
Stages in sex pilus conjugation
- Best known F pilus
- Conjugation:
1. F plasmid cell attaches itself to a cell without the F plasmid
2. Pilus retracts and fuse
3. Exhange of F plasmid
4. Both have the F plasmid
What are bacterial endospores
- Dormant stage in bacterial life cycle (metabolically inactive)
- Forms when the cell becomes vegetatively stressed
- They are extremely resistant to heat, desiccation and radiation
- Germinate when conditions become favourable
- Only found in gram ve+
What triggers sporulation
- Formation of endospores
- Complex series of differentiation events, controlled by many genes
- Triggered by nutrient depletion
Stages of sporulation
- Vegetative cell is under stress
- DNA is organised along cell axis
- Genome copy is enclosed in a forespore septum, forespore is formed
- Cell membrane engulfs forspore in a second membrane
- Spore is dehydrated making it heat and chemical resistant
- Exosporium layer are produced
- Spore matures with complete cortical layers
- Cell lyses releasing spore
Germination of endospore
- Uptake of water and amino acids trigger germination
- Re-hydration and a loss of resistance
- Cell is released and grows in the normal way
- Gram negative when released but develops and becomes positive again.
How was spontaneous generation disproved
Pasteur sterilised contents of the flask and showed the flask remained sterile as long as it was untouched
What is kosh’s postulates
That a specific microorganism causes a specific disease
- Pathogen is present in disease and absent in healthy animals
- Suspected diseased organisms are grown in pure culture
- Cells from the pure culture of the suspected diseased organism should cause disease in a healthy animal
- The new infected organism is isolated and shown to be the same as the original
Suggested benefit of the human microbiome
- Shields body tissue against invasion of “bad bugs”
- Production of vitamins from bacteria
Hard to determine what microbes cause disease and what are effected by disease
Factors that determine virulence
- Adhesion and entry into cells
- Antiphagocytic activity and immune system invasion
- Production of toxins
Why is MRSA and VRSA a threat
They have limited or no treatable antibiotics left
What are emerging “new” bacterial pathogens
“New”- not previously known as pathogens
What are the 4 major classes of pathogens
- Extracellular bacteria, parasites and fungi
- Intracellular bacteria and fungi
- Intracellular viruses
- Extracellular parasitic worms
What are the challenges faced by the immune system
- Has to protect against a variety of pathogens
- Pathogens and mutate and recombine, so the immune system must be adaptive
- Re-exposure (memory)
- Rapid division (rapid response)
- Has to distinguish between self and non-self
- Has to be tissue specific
What is antigenic drift
An accumulation of mutations within genes that code for antigen
What is antigenic shift
When two or more viruses combine to form a new subtype by having a mixture of antigens from the combined viruses
What is autoimmunity
- Immune system attacks healthy tissues and cells
- Autoimmunity results from impaired regulation of powerful immune response
What are 2 immune responses
- Innate (rapid, non specific, no memory and encoded into the germ line, dependent on family history)
- Adaptive (Highly specific, slow to adapt , has memory, somatic recombination)
What is cell mediated immunity
Defence provided by specialised cells in the blood and tissues. Relies on phagocytic cells and natural killer cells.
What is humoural immunity
Soluble phase defence by secreted proteins in bodily fluids. Relies on barriers and chemical warfare.
3 lines of innate immune system defence
- Barriers: skin, tight epithelial cells, stomach acid, mucus layers
- Cell-intrinsic responses: Pathogen induced phagocytosis and degradation of ds RNA
- Specialised proteins and cells: professional phagocytosis (e.g.neutrophils and macrophages), natural killer cells, complement system
What is the mucus layer and its function
- Made of secreted mucins and other glycoproteins
- Protects against microbial, mechanical and chemical assaults
- It is slippery so it is hard for pathogens to attach
- Contains defensins
What are defensins
- Small positively charged antimicrobial peptides which can kill or inactivate bacteria, fungi, parasites and envelope viruses.
- There are multiple defensins which have different targets
The mechanism of defensins
Hydrophobic domains enter into the core of the lipid membrane of the pathogen destabilising it, leading to cell lysis.
Why do defensins lyse pathogens but not our own epithelial surfaces
They are more active on membranes that dont contain cholesterol
What are PAMPs
Pathogen associated molecular patterns are molecules which the innate immune system recognises as pathogens.
Various classes of PAMPs
- N-formylmethionine (fmet), which is used for bacterial initiation
- Peptidoglycans from cell walls
- Bacterial flagella
- LPS from gram ve-
- Mannans, glycans and chitin from fungi
What are PAMPs recognised by
Soluble receptors in the blood (complement system) and by cellular receptors (Toll like receptors)
How does complement activation target pathogens for lysis
- Lectin pathway (pathway that binds to a sugar) or alternative pathway by pathogen surfaces cause cleaving of proenzymes resulting in a amplified proteolytic cascade to c3
- c3 cleaves c3a and c3b
- c3b binds to the pathogen membrane while c3a stimulate inflammation and attracts phagocytes and lymphocytes
- Pathogen bound c3b causes a cascade of reactions
- c9 is inserted into the membrane
- c9 pore breaches the membrane, membrane attack complex is formed
- Pathogen lysis
Toll like receptors
- Binds to pathogenic fungi and sends signals to the nucleus that result in the expression of anti-fungal defensins and inflammation
- They are found in epithelial cells, macrophages, dendritic cells and neutrophils
What are the 3 major classes of phagocytes
- Neutrophils
- Eosinophil
- Macrophage
Properties of neutrophils and what are they recruited by
- Short lived cells
- Abundant in blood
- Not present in normal healthy tissue
recruited by:
- Macrophages
- PAMPs
- Peptide fragments of cleaved components
What are granulocytes and which two phagocytes are they
- Neutrophils and eosinophils
- There cytoplasm is granular
What do macrophages remove
- Larger and longer lived than neutrophils
- Removes senscent (old), dead and damaged cells in many tissues
- Can digest microorganisms
What are eosinophils
- They work in gangs and collectively kill large parasites
- They also mediate allergic inflammatory responses.
What are the cell surface receptors on phagocytes
- TLRs
- Receptors of antibodies produced by the adaptive immune system
- Receptors for complement c3 protein
What does an active phagocyte release and induces
- Releases cytokines to attract more white blood cells
- Induces the phagocyte to engulf the pathogen into a phagosome
How is the pathogen killed once in the phagosome
- Granules fuse with the phagosome releasing lysozymes and acid hydrolases in attempt to digest the cell wall.
- Also release defensins which destabilises the membrane
- Respiratory burst allows NADPH oxidase complexes to priduce highly toxic compounds
What happens when neutrophils die
They will eject their DNA in a sticky web that traps bacteria preventing their escape from the killing frenzy
How do some pathogens survive phagocytosis
- Addition of sialic acid to capsule components avoids complement attack
- Some bacteria survive and replicate in neutrophils
- Some bacteria can neutralise actin polymerisation and therefore phagocytosis
- Some bacteria can survive in macrophages
How does inflammation aid in the killing of pathogens
- Blood vessels dilate leading to swelling and accumulation of components of the complement cascade.
- Macropages also secrete cytokines inducing chemokines that attract cytokines
How does the innate immune system recognise viruses
- Recognise CpG motifs in viral DNA
- Recognition of viral ds RNA that is an intermediate in the life cycle of viruses
What are interferons
- Cytokines that interfere with viral infection
- Induced by ds RNA
- Work in an autocrine nature, induces change in infected cells
- Works in a paracrine nature, induces change in neighbouring cells
How do interferons work
- They virally infected cells and there neighbours into less efficient factories for making new viruses:
- Warn neighbouring cells of infection and induces expression of other cytokines
- Activate ssRNA nucleases, which degrade host ssRNA, reducing protein synthesis
- Activate other mechanism that shut down host cell synthesis
- Promote apoptosis
- Upregulate expression of viral peptides of infected cells to provide recognition for activated T cells
- Stimulate expression of immunoprotoesome to process and destroy viral proteins
- Attract natural killer cells and activate macrophages
- Fights cancers
Role of natural killer cells
Cause cells with low expression of immune system recognition molecules that have been down regulated by viruses to commit suicide by apoptosis.
Describe the adaptive immune system
- Can raise a response against pathogens they have never encountered
- Highly specific
- Long lasting protection
- Response is generated by antigens
- Is recruited and trained by the innate immune system
What is the process of immunisation
- A harmless pathogen is mixed with adjuvant and is injected
- Adjuvant comprises of mycobacterial proteins and irritants which activates an innate immune response
- The innate immune response recognises the foreign antigen, which is then used to train the adaptive immune response.
Where do lymphocytes develop
The central/primary lymphoid organs:
- Bone marrow
- Thymus
Where do lymphocytes migrate after development
They migrate to the peripheral/secondary lymphoid organs:
- Lymph nodes
- Skin
- Respiratory tract
1950s experiment
- Mouse infected with antigen produced a normal immune response
- A second mouse was heavily irradiated and was unable to generate an adaptive immune response, but could still react via some innate response
- Transfer of lymphocytes into the second mouse restored adaptive immunity
This established that lymphocytes were responsible for the adaptive immune response
How do dendritic cells link the innate immune system to the adaptive system
- They are widely distributed around the body and display a variety of TLRs
- Activated dc phagocytose and beome APCs
- They then migrate to a nearby lymphoid organ and activate the adaptive immune response
What do T-cells develop from
They develop from thymocytes in the thymus, which are developed from stem cells
How are T-cells activated
- Dendritic cells present peptides to T cells
- T cell receptors recognise non self antigens
- Leads to activation, clonal expansion of specific T cells
Why do APCs only present to T cells
- Co-stimulatory molecules on the APC dock with specific co-stimulatory molecules on the T-cell
- A peptide on the APC is scanned by T cell receptor
What are the 3 types of T cells
- Helper
- Cytotoxic (killer)
- Regulatory (suppressor)
Role of T helper cells
Activates macrophages, dendritic cells, B cells and maintains cytotoxic activity by secreting a variety of cytokines
Role of T regulatory cells
Inhibit the function of T helper cells, cytotoxic cells and dendritic cells
Role of cytotoxic T cells
Kill infected host cells by persuading them to commit suicide apoptotically
2 strategies on how cytotoxic T cells kill
- Secrete porins in the cell wall. The T cell secretes specific protreases which enter the cell and activates CASPASES, the effector protein for apoptosis
- T cell bind to receptors on the target cell and sends signals that activate CAPSASES, the effector protein for apoptosis
What happens to activated B cells
They differentiate into antibody secreting effectors. There is also an increase in size of the ER for secretion of antibodies
What is the basic antibody structure
Tetrametric, 4 polypeptide chains, 2 heavy, 2 light, held together by covalent disulfide bonds between heavy and light chains
How do antibodies clump pathogens
One antibody can bind to 2 antigens. Antibodies can be cross linked due to there flexible hinge causing them to be trapped in large-cross linked networks
What are the different classes immunoglobulin (Ig) and how are they distinguished
They are distinguished by their heavy chains IgM IgD IgG IgA IgE
First antibody that a B cell makes
IgM, which will switch to other Ig molecules, but will retain the same specificity.
IgM is a pentermer of the basic triameric unit held together by J chains.
Role of IgM
- They can be secreted or be a receptor like IgD
- Can activate the complement protein and therefore considered an opsonin.
Role of IgG
- Is an opsonin for phagosytosis
- Provides passive immunity, some IgG subclasses can cross the placenta
What is an opsonin
A molecule that targets antigens for phogocytosis.
What is IgA and its roles
IgA is a dimer of two tetrameric structures held together by a J chain, and also an S chain (secretory component), which allows secretion into:
-Saliva, tears, milk and mucus.
IgA protects our mucosal surfaces: and provides some passive immunity to newborns via milk
Role of IgE
- Binds to Fc receptors on mast cells, eosinophils and basophils
- They act as passively required receptors for them
- Binding to IgE releases histomines by exocytosis, this is degranulation
The adaptivr immune system can direct parts of the innate immune system
What is class switching
Mature B cells can switch classes from IgM to other classes whilst maintaining the same specificity
What are antigen biding sites composed of
Composed of variable heavy and variable light domain interactions
Process of class switching
- Variable heavy gene is segmented from constant heavy domains of other Ig classes by introns.
- Variable heavy gene is used to maintain the same specificity
- Genomic DNA is looped and deleted, resulting in class switching.
Generation of antibody diversity
- 3 antibody genes. 1 heavy, 2 light. lambda light chain and a kappa light chain #
- There are multiple gene segments that encode the variable domains. That combined with c domains by somatic recombination.
- Variable gene segments need small pieces of diversity and joining DNA to link variable domain with the constant domain
- Affinity maturation: overtime antibodies become more specific and have improved affinity, caused by an accumulation of point mutations in the v domain.
Where does affinity maturation occur
- Occurs in germinal centres in lympth nodes.
- Here mutation rates are a million time greater
- Mutation is confined to gene segments that encode v domains. This is called somatic hypermutation.
Stages of affinity maturation
- Antigen stimulation causes activation and clonal expansion of B cells
- Some B cells proliferate in germinal centres and undergo somatic hypermutation
- Most hypermutated clones are worse and die. But rare B cells will have a high affinity for it to proliferate.
- Repeated cycles result in higher affinity antibodies.
Why is the secondary response greater and more efficient
- Memory cells
- Domiated by class switched antibodies
Different classes of T memory cells
Some carry cell surface receptors characteristic of T helper cells or T cytotoxic cells.
What is variolation
Immunisation of non-infected individuals with materia from variola infected patients
What did edward jenner find
Milkmaids infected with cowpox were immune to small pox. cowpox induced blisters offered protection.
How were viruses identified
- Infected tobacco plants could transmit disease to other tobacco plants
- Filtering extracts with ceramic filters fine enough to remove bacteria did not prevent transmission
- Postulation of a virus
Define a virus
An infective agent that consists of a nucleic acid molecule in a protein coat, able to multiple only within the living cells of a host
what is diluted by each transmission and what isnt
toxin, a virus
What is the importance of understanding historical epidemics
Can help prevent future outbreaks by antiviral therapies and vaccination programs
How do viruses affect morbidity and mortality in developing and non developing countries
-In highly developed countries
viruses cause little mortality but considerable morbidity
-In less developed countries viruses cause significant mortality
Define zoonosis
A disease that can be transmitted from animals to humans
Define morbidity
The condition of being diseased
What did viral irradication in the late 19th/ early 20th c cause
Population increase and increase in age demographic.
Life expectancy doubled
Types of viruses
- DNA
- RNA
- Double stranded
- Single stranded
- Positive sense
- Negative sense
What is the baltimore classification system and how many classes are there
- The pathway a virus takes from genome to mRNA classifies it. This is based on polarity and nature of their genomes.
- 7
Baltimore Class 1
DNA -> mRNA
Normal double stranded DNA trascription to mRNA
Baltimore class 2
(+)ssDNA -> dsDNA -> mRNA
dsDNA is an intermediate that is the template for transcription
Baltimore class 3
dsRNA -> mRNA
Can be transcribed directly and replicates via a positive ssRNA intermediate
Baltimore class 4
(+)ssRNA -> (-)ssRNA -> mRNA
Requires a negative strand template. Replicative intermediate involved.
Baltimore class 5
(-)ssRNA -> mRNA
Acts as a template for transcription. Replicative intermediate involved.
Baltimore class 6
ssRNA-RT -> DNA/RNA -> sdDNA -> mRNA
Single stranded, positive sense RNA virus replicate via dsDNA intermediate. All possess reverse transcriptase (RT)
Baltimore class 7
dsDNA-RT -> mRNA Double stranded DNA viruses that replicate via a RNA intermediate. Uses reverse transcriptase but unlike class 6 this occurs inside the virus particle on maturation.
Aims of the international committee of taxonomy of viruses
- Develop agreed taxonomy
- Develope agreed names
- Classification and nomenclature
There are different replication strategies each related to the Baltimore classification
-
6 stages of viral replication
- Attachment
- Penetration
- Uncoating/entry
- biosynthesis
- Genomic replication
- Gene expression - Assembly
- Release
Stages of attachment
- Two step process, reversible and irreversible
- Passive diffusion of virus
- Binds to specific cell receptors (determines tropism)
What is tropism and the different kinds
- Which cell type a virus infects
- Cellular tropism
- Tissue tropism
- Host tropism
Stages of entry and the different methods
- Entry by fusion and endocytosis
- Receptors (or environmental changes) trigger irreversible conformational changes. Environmental changes can be a change in pH.
- Conformational changes allow entry/release of genetic material
Stages of viral replication and biosynthesis
- Negative sense strands used as a template for mRNA
- Positive sense strand can be directly translated; template for -ve strands
- Polyprotein processing
Stages of assembly
- Formation of structural units of the protein shell, can be from one or several viral proteins.
- Assembly of protein shell by interactions among structural units
- Selective packaging of nucleic acid genome and other viral components
- May be formation of an envelope
Two different methods of viral release
- Lysis: viral load exceed a critical level, the cell bursts and new viruses are released
- Budding: Viral capsids become enveloped and buds off
Define pathogenesis
Mode of development of a disease, the mechanism by which a pathogen causes a disease
Define pathogenicity
Nature and severity of the diseased caused
Define virulence
Refers to different pathogencicity of different strains of the same virus
Easiest way for a virus to become systemic
Enter lymphatic or circulatory system
Viral factors that affect pathogencicity
- Nature of target tissue
- Ability for the virus to replicate and disseminate faster than the immune system develops
- Virus evading the immune response
- Severity of inflammatory response
- Extent of adaptive response
Host factors to pathogenicity
- Prior experience of infections
- Immunosuppression due to other diseases
- Intrinsic features of immune system (e.g. gender differences)
- Physiological variation (fitness, age, stress)
A major cause of enhanced susceptibility
Malnutrition - through reduced cellular immunity. This accounts high mortality rates in developing countries.
Other factors affecting pathogenicity
- Size of dose
- Climate effects
- Circadian effects
- Socio-economic factors (e.g. alcohol)
3 outcomes of infection
- Acute infection, followed by death
- Acute infection, followed by recovery
- Persistence infection with continuous or sporadic virus production with or without chronic disease
What is acute infection
- Short lived and intense
- Usually recovered patients is permanently immune, but re-infection may occur
- e.g. measles, ebola
When does re-infection occur
When there is variation in epitopes between different strains of a virus so that previous antibodies cannot neutralise current virus of a different strain.
What is an epitope
Part of an antigen molecule to which an antibody attaches to.
What is persistent infection
“continuing in existence”
- Chronic: constant virus production
- Latent: usually no detectable virus but production may re-activate
- Natural immune response fails to clear virus
- Symptoms may be on-going, develop later
- Re-activation by immune suppression
Immune invasion in persistent infection
All viruses have evolved functions that help evade the immune response. They tend to do little damage.
3 broad viral disease control strategies
- Interrupt transmission
- Protection of vaccination
- Treatment of infection
Horizontal transmission of viruses and its different routes
- Host to host (direct or indirect contact)
- Respiratory
- Oral faecal
- Sexual
- Saliva/urine
- Mechanical: skin puncture
- Conjunctival: into eye
- Fomites
Factors affecting respiratory transmission (airborne)
- Aerosol droplet size
- Humidity
- Seasonal factors
Factors affecting oral faecal route (contaminate food/water)
- Handwashing
- Water supply
- Quality of sanitation
- Weather e.g. rainfall can overwhelm sewage systems
Factor affecting sexual transmission
- Number of partners
- Sexual orientation
- Gender
Saliva and urine transmission
-
- Extension of respiratory route
- Salivia and urine get into aerosol which can be inhaled
- Urine transmits sterile viruses that replicate in the kidney
Fomite, skin and conjunctiva
- Fomites: solid objects been om contact with secretion and excretion
- Skin: sever skin damage allows systemic access, may target basal layer (warts)
- Conjunctiva: hand to eye contact
What is indirect transmission
Involving one or more other species
- Vectors transmit viruses between humans
- Viruses may be harboured by another reservoir species and transmitted to humans (zoonosis)
- Insect borne viruses
What is vertical transmission
Mother to child
- Intrauterine: viruses cross the placenta
- Perinatal: transmission during delivery
- Postnatal: breast feeding
How to interrupt transmission
- Personal behaviour (e.g. hygiene, mosquito net, condoms)
- Public education campaigns
2 key points of vaccinations
- Infection leads to life long immunity
- Low virulence strain may lead to resistance to a high virulence strain
What is a vaccination
A means of inducing immunity without the accompanying disease
2 types of vaccine
- Live but attenuated, which infect and replicate but cause little or no disease
- Killed, non-replicating but capable of inducing immunity, may be biochemically purified or genetically engineered
How are live attenuated viruses obtained
- Attenuation by serial passage where wild type virus is inoculated into heterologous (wrong) host
- After the virus has replicated it is taken from the first host and inculcated into a second
- Growth in the wrong hosts selects variants which are no longer virulent but still replicate, in the autologous (usual) host
Problems with live attenuated viruses
- Cannot predict how many passages are needed
- Cannot predict if will ever work
- We have no understanding how it works
Inactivation process of killed vaccines
- Destroy all viral infectivity
- Dont destroy antigenicity of viral proteins
What is needed for a vaccination program to be effective
Cheap and safe
Basic Ebola facts
- Linear, negative sense single-stranded RNA virus
- Part of the filoviridae family
- Reservoir host is bats
- Transmission by direct contact with contaminated bodily fluids
Ebola outbreak 2013
- Affecting west Africa in december 2013, significant deaths
- Epidemic was not identified until months later
- People could cross borders
- Was declared a public health emergency of international concern by WHO
3 types of influenza based of capsid protein
- Type A: serious pandemics in humans and other animals
- Type A and B: seasonal epidemics
- Type C: Mild human respiratory illness
How do different subtypes of influenza vary
In protein spikes
- 18 different hemagglutinin (H) which aids in viral entry
- 11 different Neuraminidase (N), which aids in viral exit
What is antigenic drift in influenza
- An accumulation of mutations in hemagglutinin and neuraminidase genes
- Viruses dont have proof reading mechanisms and cant correct mutations
- Causes seasonal influenza epidemics
What is antigenic shift
- 2 different viruses combine when they infect the same cell
- Gene segments are exchanged during replication
- They exchange H and a new mosaic virus is created
5 techniques to study viruses
- Virus culture systems
- Plaque assays
- Haemagglutination assay
- Antibody detection
- Molecular mechanism
How do virus culture systems work
- Observation of disease generation in an organism
- Identification of causative agent using EM
- Cultured in a lab to amplify and then study its effects on cells or an organism
3 main culture systems
- Animal: natural infection but is variation between individuals and there are ethical concerns
- Organ culture: natural infection, however tissue is not subject to normal homeostatic mechanisms
- Cell culture: clones so less variation and environment can be easily manipulated
Different cell lines for the cell culture
- Primary cell lines: differentiated but limited to a few rounds of growth
- Cell lines: less differentiated, diploid survive > 50 passages
- Continuous cell lines: dedifferentiated but immortal (HeLa cells)
What is the cytopathic effect
Viral infection may change the phenotypic appearance of cultured cells
Cell culture problems
- Viral entry is dependent on cell surface receptors
- Not all cells can be cultured
- Rapid viral replication can lead to viral mutation
What is a pathogencicity assay
- Dilute virus stock and infect 4 animals per dilution
- At dilution infecting half (1/2), there is one AID50 unit
What is a plaque assay
- Virus is added to a monolayer of cultured cells at each dilution
- Then covered with semi solid agar
- Then incubated and stained with crystal violet
- Sites of infection leave a plaque which appears white against the blue background
1 plaque = 1 PFU (plaque forming unit)
How does a haemagglutination assay work
- Some viruses can bind to red blood cells
- They cross link the erythrocytes
- The virus erythrocyte complex can be used to quantify the virus as it leaves the sample in a bound complex
- If there is no reaction, or the virus has been inhibited red blood cells will sink to the bottom of the well. A dot will appear.
1 HA unit is found at the end point.
2 methods of detection for viruses using monoclonal antibodies
- Immunofluorescence (IMF)
- Enzyme linked immunosorbent assay (ELISA)
How does immunofluorescence (IMF) work
- Antibodies specific for viral proteins
- Add a fluorescent tag
- Allows visualisation of infected cells, however you cant quantify viral load.
3 types of ELISA
- Sandwich ELISA
- Indirect ELISA
- Direct ELISA
How does sandwich ELISA work
- A captured antibody is bound to the bottom of the well and a viral antigen will bind
- Primary antibody will bind to antigen
- A secondary antibody from a different species binds to the primary antibody
- Secondary antibody has a conjugated chromogen which cleaves a substrate causing a colour change
How does indirect ELISA work
- Antigen is bound to the bottom of the well
- Primary antibody will bind to antigen
- Secondary antibody from a different species binds to the primary
- Second antibody has a conjugated chromogen and causes a colour change
How does direct ELISA work
-Primary antibody binds to antigen with conjugated chromogen to cause a colour change.
How to quantify colour change in ELISA
A standard curve is used to find the number of viral proteins
Molecular techniques in detecting viral nucleic acids
- PCR
- Real time PCR
PCR in detecting viral nucleic acids
- DNA is extracted
- DNA is replicated using specific primers, which will bind to a specific site
- Run through electrophoresis
- Compare bands to viral DNA
Real time PCR: Quantitative measure of viral load (RNA viruses only)
- Extract RNA from virus
- Convert RNA to DNA
- Measure how long it takes to produce a certain amount of DNA
- The more cycles it takes the more dilute the virus
What are characteristics of microparasites and examples
Small, difficult to count and multiply in their host
- Viruses
- Bacteria
- Fungi
- Protozoa
What are characteristics of macroparasites and examples
Large, can be counted, multiply eternal of the host
- Endoparasites (worms)
- Ectoparasites (ticks, flees, lice)
Different transmission routes
One to one contact:
- Direct (physical contact)
- Indirect (fomite)
- Droplet (contact through air, large droplets)
Non-contact:
- Airborne (transmission via aerosols, small particles)
- Vehicle (A single contaminated source e.g. food/water)
- Vector (By insect or animal vector)
What is disability adjusted life years (DALY)
The number of healthy years of life lost due to premature death and disability.
DALY = years lived with disability + years of life lost
Plague Fact file
- Caused by Yersinia pestis
- Endemic in nature
- Transmitted by fleas with rodents as hosts
- 3 subtypes of disease
- High mortality without treatment
3 subtypes of the plague
- Bubonic: Infection of the lymphatic system
- Septicaemic: Infection in the blood
- Pneunomic: Infection in the lung
Chagas fact file
- Caused by Trypanosama cruzi
- Transmitted by triatomine bugs when taking a blood meal
- Acute disease characterised by inflammation and acute myocarditis
- Chronic disease characterised by kidney disease and chronic myocarditis
Define infection
Presence of the pathogen/parasite in the host
Define disease
clinical state of host
Macroparasites nature/characteristics
- Chronic disorders
- Endemic in nature
- There is limited post-recovery immunity
- High percentage of re-infection
- Multiple worms in infected individuals
Life cycle of soil-transmitted helminths and schistosomiasis
- Predominantly oral / faecal transmission
- Most “adult” macroparasites reside in GIT
- Eggs released in faecal matter
- Behaviour-related exposure
Define epidemic
An increase in the incidence of disease in excess of that expected
Define incidence
Number of new cases per unit time
Define R0 and its equation
The average number of new cases arising from one infectious case introduced into a population of wholly susceptible individuals
R0 = PxCxD
R0 = 1+L/A
L- life expectancy
A-average age of infection
What is Re
Effective R, the true repoductive rate.
Re = R0 x fraction of susceptible individuals
An epidemic ends when Re declines to < 1, until new susceptibles are generated.
What causes and epidemic to continue
- Susceptibles increase through births or migration
- Pathogen mutates and can re-infect individuals
- No immunity
- Immunity wanes
What is an epidemic fade out
The elimination of the infectious agent due to chance. This is more common in smaller populations.
What is waning immunity
Loss of immunity post recovery from infection
What can we learn from patterns in epidemic data
- Prevalence and incidence
- Origin of outbreak
- Mode of spread
- Potential incubation and time of exposure
Define incubation period
The period between infection and clinical offset of disease
Define latent period
The time from infection to infectiousness
What is a point epidemic
- Single common exposure and incubation period
- Doesn’t spread by host to host transmission
- Everyone gets sick in a short period of time
- e.g. food borne disease outbreaks
What is a continuous source epidemic
- Prolonged exposure to source overtime
- Cases do not occur within the span of a single incubation period
- Curve decay, may be sharp or gradual
- e.g. cholera
What is propagated progressive source epidemic
- Spread between hosts
- Large curves until susceptibles are depleted, or intervention is made
Cholera facts
- Caused by Vibro cholerae
- Cholera is a microparasite, infecting the small intestines
- Cholera toxin inhibits water absorption
What is endemic equilibrium
- There is stability in the incidence of infection (constant).
- Parasite is persistent in host population
- Re=1
How to calculate fraction of susceptibles (s)
1/R0
What are the determinants of persistence
- Critical community size
- Rate of contact (mixing) for transmission
- Duration of infectious period
- Survival of host
Define critical community size (ccs)
The minimum host population size required for the pathogen to persist. A bigger concern for microparasites.
The effect of increasing infectious period but retainin R0
It eliminates cycles, improves persistence and increases incidence.
Ebola fact sheet
- Zoonotic pathogen
- Transmitted animal to human
- Fruit bat is the reservoir host
- Human to human outbreak will cause a large outbreak
- Almost certainly there is constant human cases for ebola in isolated communities
Phocine distemper (PDV)
- Disease in grey seals
- Harp seal is reservoir host
- CCS us below in grey seals, but there is re-infection, so there must be a reservoir host
- Harp seals migrate and trigger disease outbreaks in grey seals
Define reservoir host
-A population or species that is clinically normal (not diseased) but infectious
Measurements of macroparasite infections
- Mean burden: mean number of worms per individual
- Prevalence
Summary of soil transmitted helmiths
- Host density (CCS) is not limiting factor to transmission
- External “reservoir” of transmission stages
- Long generation time and period of infectiousness
- Immunity is transient (dependent on parasite burden)
- Continual re-infection
- Mode of transmission: often contaminative – not requiring host to host transmission
Define control in epidemiology
Maintaining the parasite population to an acceptable level
Define elimination
Zero incidence in a defined geographical area
Define Eradication
Zero incidence worldwide
Define extinction
Infectious agent no longer exists in nature or in a lab
How to prevent transmission
- Mass (random) or targeted vaccination
- Vaccination by risk group
- Spatial vaccination (ring vaccination)
- Reduction in contact
What happens in intervention after transmission and what is the aim of intervention
Infectiousness curtailment - tracing, then isolation or culling.
To reduce the number susceptible, reduce time infectious and reduce contact.
Define immune proportion (Pc) and how is it calculated
The minimum proportion of individuals you need to vaccinate for herd immunity.
pc = 1 - 1/R0
What is anthroponotic transmission
Human -> Arthropod -> Human
What is zoonotic transmission
Animal -> Arthropod -> Human
Define vectorial capacity (c)
The average number of potentially infective bites that will be delivered by all the vectors feeding upon a single host in 1 day.
R0 = cxd c-vectorial capacity
d-duration of host infectiousness (days)
What is vectorial capacity sensitive to
- Vector biting rate per day
- Proportion of blood meals taken on host
- Daily vector survival rate
- Latent period of the agent inside the vector
Examples of vector control
- Human bait bite traps e.g. insecticide treated net
- Non-human bait traps: lure them in without humans and kill them with insecticide
- Rural drainage of breeding sites
How does climate change effect disease
Brings extreme weather events which effect vector borne and water borne diseases.
Why are vector borne disease affected by climate change
- Environmental parasite stages are sensitive to climate
- Vectors are sensitive to climate thus affecting parasite/pathogen
e. g. seasonality in vector abundance and breeding sites - Parasite development is often climate dependent
vectorial capacity equation
((v/N)(ah)^2(p)^n)/-lnp
Define extrinsic incubation period (EIP)
The time between gaining an infectious agent by a vector and the ability of being able to pass it on
What is Dv and how do you calculate it
The time the average vector may be infectious
Dv= (p^EIP)/-lnp
What is el nino and how does it affect dengue
The warming of sea surface temperatures that occur every few years in the pacific. Has a positive correlation with dengue.
What is blue tongue virus (BTV)
- Double stranded RNA virus in livestock
- Cows are reservoir hosts
- Disease in sheep/deer
- Vector borne in midges
What are the potential biological influences in BTV transmission
Temperature
- Warm/hot periods in autumn/ summer increase transmission potentially
- Warm nights/winter increases virus persistence to “overwinter”
Precipitation
-Govern size/persistence of semi-aquatic breeding sites -Moisture govern key microhabitats for adults
What happens to the overwinter
- Vectors transfer the infectious agent to other midges
- Adult midges survive
- Persists in the host
- Vertical infection to lambs
Challenges of predicting infectious disease with climate change
- Pathogen interacts with climate as well, not just the host
- Herd immunity and public health capacity is hard to take into account
What is the Devil facial tumor disease (DFTD)
- An agressive cancer in Tasmanian devils
- 100% fatal
- Small lesions and lumps develop into tumours
- Normally on the head, but can metastasise around the body
- Death is cause by inability to eat or organ failure
How do Tasmanian devils transfer DFTD
Cancer is transferred when biting each other during fights, when mating or feeding.
What is the allograft theory
The cancer cells themselves are the etiological agent. Tumour cells are on the teeth.
Why do Tasmanian devils have no immunity
- All tumours are identical
- Genetic diversity is very low (genetic bottlenecking)
- They have a low major histocompatability complex (MHC) class 1 diversity. They have the same MHC1 response.
- DFTD cells lose expression of MHC class 1 on the cell surface. Devils don’t recognise the tumour cells as foreign, so no immune response.
- High prevalence
Ecoligical impacts of the Tasmanian devil
- Top predator: key stone species
- Its decline will alter ecosystem balance: will advantage feral predators, and scavengers
How to save the Tasmanian Devils
- Vaccine development
- Captive breeding
- Translocation: colonising a new area with healthy devils
Define prevalence
-The proportion of a particular population found to be affected by the infectious agent