Rabies and Lyssaviruses: Week 10 Notes PDF

**Wek 10 \| Rabies and Lyssaviruses** **RABIES: THE DISEASE**   If a dog is m.d and the authorities have brought the to the knowledge Of its OMterS; if he does not keep it in, it bites a man and Caused his thon the Shau pay two thirds Of a mina (40 s h eke\'s) in it bites a and causes his death h...

**Wek 10 \| Rabies and Lyssaviruses** **RABIES: THE DISEASE**   If a dog is m.d and the authorities have brought the to the knowledge Of its OMterS; if he does not keep it in, it bites a man and Caused his thon the Shau pay two thirds Of a mina (40 s h eke\'s) in it bites a and causes his death he Shan pay fifteen Shekels Of Silver. Eschunna Code of Mescoatamia century BC) (p reurscy to the Code o\' Ham murabi)   RABIES: SYMPOTOMS      **EARLY** Bite or scratch No symptoms (20-90 days)   **SYMPTOMS BEGIN** Kills \~100% once symptoms begin Sensation at original bite site Flu-like symptoms   **CLINICAL (Furious (80%), Paralytic (20%))** Fever, mouth salivates, convulsions **Hydrophobia** (unique to rabies) - fear of water Hallucinations Hypersexual behaviour Moments of clarity (come back to being normal - but only temporarily) Coma, death   VACCINE AND TREATMENT   Vaccine developed by Louis Pasteur and Pierre Roux (1885) Weaken a virulent rabies virus by aging and drying spinal cords of rabies-infected rabbits and using that to inoculate people. drying weakens the virus Joseph Meister, 9 year old boy   Vaccines improved (inactivated) Recombinant vaccines (G-protein) At risk individuals (high-risk occupations) Can be applied post-exposure (before symptoms) Rabies immunoglobulin, 5 vaccination course   RABIES: STILL A PROBLEM   \>55,000 deaths per year   Under reported, under-served, poorly resourced regions, rural. Vaccine expensive, multiple courses **Zoonotic** -- almost all warm-blooded animals can be infected. Bats, dogs, foxes, raccoons, skunks etc. \>99% human cases from dogs Almost impossible to completely eliminate due to so many wild-life reservoirs Control: vaccination of dogs, pets Wild-life oral baiting (herd immunity) Humans are a dead-end host (**no human-to-human transmission**).   RABIES IS CAUSED BY LYSSAVIRUSES (NOT JUST RABIES VIRUS)   Lyssa = Greek spirit for mad rage, frenzy At least 14 members of the Lyssavirus genus Rabies disease is not caused only by rabies virus These other diseases can also cause rabies-like diseases   A diagram of different types of bats Description automatically generated   DOES AUSTRALIA HAVE RABIES?   **Australian Bat Lyssavirus Virus (ABLV)**   Another virus that causes rabies disease.   Very similar to rabies virus Zoonotic -- flying foxes, bats 3 human cases recorded so far, 3 fatal 1996: an animal carer bitten and died within 8 weeks. 1996: a woman was bitten, 2 years later she died. 2013: 8yo boy dies, bitten by bat 2018: Hawthorn man bitten by bat carrying ABLV (not fatal).   **Treatment**: Same as for rabies, rabies immunoglobulin and rabies [vaccine] protects against Lyssavirus **Prevention**: Avoid handling bats.   RABIES INFECTION OF THE HOST      1\. Animal bite or scratch (virus in saliva) 2\. Infects muscle (replicates), transmitted to peripheral nerves, then central nervous system (CNS) 3\. Virus particles transport in along neuronal axons (i.e. **retrograde** = towards cell body) in vesicles using microtubules. 4\. When at neuronal cell body, released from vesicle, replicate, assemble new virus particles, then infect next neuron. 5\. Travels up spinal cord, leading to brain, causes **encephalitis**. 6\. Spreads to other organs (e.g. Salivary glands)     A diagram of a cell membrane Description automatically generated     Gets endocytosed into the neuron Goes down microtubules toward cell body Goes and keeps going from neuron to neuron infecting them all   Retrograde axonal transport. Slow process (depending where bitten) -- allows time to vaccinate! Very little sign of damage to neurons (how does it kill?) Very 'stealthy' virus -- very little cytopathic effect Inhibits apoptosis Immunosuppressive strategies (immune evasion)   THE CENTRAL DOGMA \... VIRUSES DONT CARE      \*humans have blue   POSITIVE AND NEGATIVE SENSE RNA VIRUSES   e, g, virus posmvE SENSE RNA CYTOPLASM UCLE P r OtOin eg. Viral RdRp REPLICATION Rabies vims NEGATIVE SENSE RNA c YTOPLASM REPLICATION Viral Rd Rp protein MtJST MAKE OWN RNA POLYMERASE\' MUST BRING AND MAKE OWN RNA POLYMERASE!   \*positive sense: mRNA, its ready to make proteins   \*negative sense: it needs something to make the +RNA therefore it needs to bring the viral RdRp. Once the +RNA is acquired, the host cell\'s ribosome can be used to make the protein.   LYSSAVIRUSES: THE STATS        **It\'s an RNA virus**: which means it mutates quickly - because their polymerase (RdRp) doesn\'t have proofreading ability like our DNA polymerase. As a result, they make more errors and mutate more quickly.   Single-stranded negative sense: needs to bring its own RdRp.   One segment: one molecule/one piece of RNA   Mononegaviruses: \"mono\" one segment \"neg\" negative sense viruses   **RABIES: THE INFECTIOUS CYCLE**   RABIES VIRION/PARTICLE   10 (M) Nuclăproteln PC\" PhOSp oprOten (P)   **Glycoprotein**: essential for binding to the protein and getting in. **Matrix protein**: required for assembly.   Enveloped (from host cell plasma membrane) Helical All 5 proteins in particle (N, P, M, G, L) N binds and covers (encapsidates) RNA genome (nucleoprotein) N protein binds to the RNA every 9 base pairs and wraps it up into a helix. G on surface (spikes)      LYSSAVIRUS GENOME   --- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------   (-) RNA GENOME 3\' mRNA (+) Protein Nucleoprotein (N) Phosphoprotein (P) Matrix Glycoprotein (G) Large protein (L) ---11-12 kbp RNA-dependent RNA Polymeraæ (L) Ribosomes (host) They don\'t waste space! \> encapg&tæ gme for replication) z co-factor o\' polymerase L, links L to N RNA. required for transcription replication: binds N, immune -s required tor viral assembly and budding of \*Nicle, requ\*ed into cell \> RNA-dependent RNA polymerase (RdRp), --- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------   The virus uses the host\'s ribosomes to make proteins.   RABIES/LYSSAVIRUS INFECTIOUS CYCLE   **Attachment and Entry**   Rabies virus enters by **receptor-mediated endocytosis**      1. The virus has the glycoprotein on the outside which is responsible for binding to the surface. 2. This induces for endocytosis to occur and form an **endosome**. 3. The endosome hooks onto microtubules and gets transported towards the cell body.     **Getting particle out and into the cell:**   су---т   1. pH inside the endosome becomes more acidic \--\> causes conformational change in glycoproteins. 2. The shape changes and fuses into endosomal membrane (viral and endosome membrane fuse) 3. Nucleocapsid (rna with nucleoprotein wrapped around it - L and P protein) is released   **Synthesis**   Nucleocapsid is released into cytoplasm   Nucleocapsid is a **Ribonucleoprotein (RNP)** -- complex composed of RNA and proteins. (in this case, the P, N and L protein)      Need to bring N, P and L for RNA synthesis   A diagram of a virus Description automatically generated   **Transcription (mRNA synthesis)**        **P protein** is a co-factor for the viral RNA polymerase (**L**). P binds N-RNA and L (links them together). - [VERY IMPORTANT] Transcriptase uses **start-stop mechanism** -- specific signals between genes, disengages from template, re-engages. Generates mRNA with cap and poly-A tail (generated by L protein).   L (-) RN L L TRANSCRIPTION aHA01ENT   Sometimes the PNL complex can fall off then the transcription has to start again to make the mRNA. Because of this, more of the start mRNAs get made then the end ones (e.g. more N and P get made than G)   **Transcription gradient** generated due to stop-start mechanism -- polymerase complex doesn't always re-engage with genome (N \> P \> M \> G \> L)   Rabies virus generates multiple isoforms of the phosphoprotein (P) by the process of **ribosomal leaky scanning**. This is when the ribosome fails to initiate translation at certain start codons due to weak kozak sequences, causing initiation of translation to occur at downstream start codons.   **Translation**   To make the protein from the mRNA.      **Replication**   A diagram of a diagram Description automatically generated with medium confidence   Genome (-RNA) makes antigenomes (+RNA) in order to make new genomes. Replication requires new N protein to encapsidate RNA (i.e. translation of viral proteins) If no viral translation = no trigger to replicating the genome (just transcription of mRNA)   **Replication occurs in \"liquid\" Negri Bodies**   First described by Adelchi Negri in 1903. **Negri Bodies**: Membrane-less cytoplasmic inclusions caused by rabies infection. Rabies diagnostic marker Site of replication Recently shown to be liquid organelles Replication occurs in Negri bodies -- formed by N and P Likely most negative-sense RNA viruses use liquid organelles   **Assembly and Release**   Assembly at plasma membrane   Three main components assembled: 1\. **Nucleocapsid** (RNA, N, P, L) - red 2\. **M** (at plasma membrane) - green 3\. **G** (glycosylated -- sugar groups added) - purple   M protein mediates assembly, 'selects' nucleocapsids (complete, not antigenome). Triggers budding from the host cell. Gain membrane envelope from host cell as buds from cell.        SUMMARY: LYSSAVIRUS INFECTIOUS CYCLE   A diagram of a cell Description automatically generated   1\. G binds cellular receptor and endocytosed into endosomes. 2\. Low pH of endosome causes conformational change in G, triggering fusion with endosomal membrane, releases of viral RNP into cytoplasm (nucleocapsid, N, P, L). 3\. Transcription occurs (requires viral proteins), making mRNA Transcription stop-start gradient causes different levels of viral mRNA. 4\. mRNA translated by ribosomes 5\. G protein translated into ER/secretory pathway 6\. Replication begins when enough N is made, N coats new viral RNA (make antigenomes) 7\. Replication occurs in Negri Bodies in the cytoplasm. 8\. Antigenomes make new viral genomes.   New genomes used for: 8\. Transcription 6\. Make more antigenomes 12\. Packaging into new particles.   Viral genomes are packaged at the plasma membrane. 11 & 12. M associated with membrane, packages genomes into particles.   13\. G passed through secretory pathway, glycosylated (9 and 10) at the plasma membrane packaged on outside of particle, and particles released from cell by budding from membrane (gain envelope). IMMUNE RESPONSES   **Cytokines** = small proteins secreted by cells, involved in signalling/communicating with other cells. E.g. **interferon**.      INNATE IMMUNE DEFNESES: CYTOKINES   Viruses must counteract the innate immune response.   --- -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------   AUTOCRINE same cell PAMP = pathogen- associated molecular patterns (e.g. LPS, peptidoglycan, dsRNA) PRR = pattern recognition receptors (e.g. RIG-I) affects Cytok i (e.g. interferon) PA p (active ANTIVIRAL DEFENCES!! ANTIVIRAL DEFENCES!! PARACRINE = affects different cell ANTIVIRAL DEFENCES\" --- -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------   INTERFERONS (IFN)        TYPE I INTERFERON   Three main types of IFN: **Type I (IFNα and IFNβ)** -- direct response to infection broad cellular expression and receptors Type II (IFN[*γ*]{.math.inline}) -- immune cells Type III (IFN[*λ*]{.math.inline})   Production of IFNα/β is rapid: within hours of infection, declines by 10 h IFN binding to IFN receptors leads to synthesis of \>1000 cell proteins **(IFN stimulated genes = ISGs)** Many antiviral Mechanisms of most ISGs not known   IFN INDUCTION AND SIGNALLING   RIG-I = retinoic acid-inducible gene I IRF3 = Interferon regulatory factor 3 IFNAR = Interferon Type I receptor STAT = signal transducer and activator of transcription ISGs = interferon stimulated genes   INDUCTION mRNA Type \'FN     Virus releases its genome into the cell RIG-I (PRR), which is normally inactive in the cell, picks up the signal and triggers it to become activated causing the whole signal transduction of events to occur IRF 3 (regulatory factor) gets phosphorylated to form a dimer It can then get transported into the nucleus \... **1. Recognition of Pathogen-Associated Molecular Patterns (PAMPs):** - **Pattern Recognition Receptors (PRRs):** Cells detect the presence of viral infection through PRRs like RIG-I. - **Activation:** Upon binding to PAMPs, RIG-I becomes activated. **2. Signal Transduction:** - **Downstream Signaling:** Activated RIG-I triggers a signaling cascade involving various proteins, including IRF3 and TBK1. - **Phosphorylation:** IRF3 is phosphorylated, leading to its activation. **3. IFN Production:** - **Nuclear Translocation:** Activated IRF3 translocates to the nucleus. - **Gene Transcription:** IRF3 binds to specific DNA sequences, promoting the transcription of type I IFN genes. - **mRNA Synthesis and Translation:** The transcribed genes produce IFN mRNA, which is then translated into IFN proteins. **4. IFN Signaling:** - **Binding to Receptor:** IFN proteins bind to their specific receptors (IFNAR) on the surface of neighboring cells. - **Signal Transduction:** Binding to IFNAR activates a signaling cascade involving STAT1 and STAT2 proteins. - **Nuclear Translocation:** Activated STAT proteins dimerize and translocate to the nucleus. **5. Antiviral Gene Expression:** - **Gene Transcription:** STAT proteins bind to specific DNA sequences, promoting the transcription of antiviral genes. - **mRNA Synthesis and Translation:** The transcribed genes produce mRNA for antiviral proteins, which are then translated. **6. Antiviral Effects:** - **Interferon-Stimulated Genes (ISGs):** The produced antiviral proteins, known as ISGs, have various functions to inhibit viral replication. - **Antiviral Activity:** ISGs can block viral entry, replication, and protein synthesis, helping to limit viral spread.  VIRUS ENCODE IFN ANTAGONISTS      Three broad strategies 1\. General inhibition of host gene expression (red) E.g. Shutting down translation; iFN expression etc. 2\. Sequestration/masking of PAMPs (green) 3\. Sequestration/modification of signalling components (pink)   Often multiple strategies IFN antagonists often multifunctional viral proteins.   VIRUSES USE MANY WAYS TO DISRUPT IFN PATHWAY   卜 - OENV NS-S EBOV VP\'S   Basically you can stop the switching on of interferons   RABIES ANTAGONISE THE IFN RESPONSE      Rabies must counteract the hosts innate IFN response. At least one protein must act as an IFN antagonist.   RABIES P-PROTEIN BLOCKS IFN INDUCTION AND SIGNALLING   SIGNALING A binds p-protein inhibits phosphorylation of STATI,Q, prevents nuclear import Blocks activatön of ISGs likely using IRF3, therefore IRF3 cannot be activated Mechanism not clear tea   RABIES P-PROTEIN: A MULTIFUNCTIONAL PROTEIN      Binds L and N protein for transcription and replication C terminus binds N protein N terminus binds L protein CTD binds many host proteins (many unresolved functions) CTD binds phosphorylated STAT1/2 Traffics between nucleus and cytoplasm NLS = nuclear localisation sequence (triggers it to be imported into the nucleus) NES = nuclear export sequence   RABIES P-PROTEIN: MULTIPLE WAYS TO INHIBIT IFN SINGALLING   --- --------------------------------------------------------------------------------------------   +1FN p transc 1 P binds and prevents nuclear 2. P traffÆ into nucleus and brings STATS out --- --------------------------------------------------------------------------------------------   P only binds when STATs are phosphorylated (i.e. active) and stops it from getting into the nucleus to switch on the interferon stimulated genes   RIBOAOMAL LEAKY SCANNING        **Kozak Sequence** = nucleotides around start codon determines likelihood the ribosome initiates translation Weak Kozak = can skip AUG Strong Kozak = will start at AUG   P PROTEIN ISOFORMS: 1 GENE, MANY PROTEINS   A screenshot of a computer screen Description automatically generated   RABIES P-PROTEIN: MULTIPLE WAYS TO INHIBIT IFN SIGNALLING   --- -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------    --- -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------   P-PROTEIN INHIBITION OF STATS IS CRITICAL TO PATHOGENESIS   Path Rabies 100 passages in Rabies Pathogenesis associated with P-protein Attenuated (-0 at hogenic P)      RABIES IFN ANTAGONISM: SUMMARY   Rabies effectively impairs the host IFN response P-protein is the primary IFN antagonist P-protein IFN antagonism is multipronged: Impairs IFN induction (mechanism unclear) Inhibits phosphorylation of IRF3 (mechanism unclear) Impairs IFN signalling (multiple ways) Binds phosphorylated STAT proteins Many ways to prevent transcription of ISGs. P-STAT interaction associated with pathogenesis   **RABIES: USING FOR GOOD**   THE MANY \'TRICKS\' OF RNA VIRUSES   RNA viruses have small genomes Use clever 'tricks' to increase coding capacity   These include: Ribosomal leaky scanning 1 RNA -\> polyprotein, cleaved Multifunctional proteins Ribosome frameshifting Suppression of termination Proteins regulated (trafficking signals, conformational, PTM, etc)   Viruses teach us about what is possible and provide useful tools   HOW TO MANIPULATE RABIES VIRUS?   Use Reverse genetics DNA copy Of RNA virus genome Insert DNA copy ot RNA genome into DNA plasmid Easy to manipulate --- mutate, insert gene. delete etc. POSITIVE STRAND RNA VIRUS easy! RNA ViMgenme ma mid (DNA) Re I e ase CYTOPLASM RNA (mRNA) Pmteim Assembly      MANIPULATING RABIES GENOME: INFINITE POSSIBILITIES   ○ + ○ + : NO   E.g. By inserting a gene for fluorescent green.      TURNING THE TABLES: USING RABIES FOR GOOD   **Neurotracer** Map the neural network (strictly trans-synaptic) using the green dye attached to it Label virus (e.g. GFP) Replicates, therefore labels each neuron similarly Low cytopathic effects   **Pass blood-brain barrier (BBB)** Rabies passes BBB by binding receptor on nerves (G protein) Found 28-residue peptide of G when fused to cargo, delivers to brain Treat West Nile Virus   **Vector for Vaccines** Use highly attenuated rabies virus Engineered to express immunogenic proteins of other viruses (e.g. Ebola, Marburg, Hendra) Very little seropositivity to rabies in population Highly immunogenic   **Curing Alzheimer's ??** Neuroinflammation major factor for many diseases (Alzheimer's, stroke etc) Rabies P is exceptional at impairing immune/inflammatory response Can we use P to learn how to target these pathways to tackle these disorders?   RABIES VIRUS: JUST ONE VIRUS   Most lethal viral disease Only 5 genes, can cause so much damage. Teaches us clever to ways to manipulate cells, and useful tools And this is just one virus!   (м) (Р)     FENDAMENTAL STEPS OF MONONEGAVIRUSES CONSERVED   All mononegaviruses have similar genome organisation   MAJOR CONCEPTS Viruses must encode IFN antagonists to counteract host innate IFN response. Viruses often use multiple (diverse) ways to antagonise the IFN response. RNA viruses have developed clever strategies to very effectively increase the coding capacity of their small genomes. The fundamental steps of the infectious cycle are conserved between all Mononegaviruses. By understanding how viruses work, we also learn about the cell, and discover new tools that can be applied to many applications.   **Week 11 \| Clostridial Infections** GRAM POSITIVE RODS   **Clostridium** - produces spores, strict anaerobes   CHARACTERISTICS OF THE GENUS CLOSTRIDIUM   Gram positive rods Obligate anaerobes; cannot grow in presence of oxygen Form heat resistant endospores; spores produced inside the cell Common inhabitants of the gastrointestinal tract Important in agriculture (soil maintenance) and industry (biofuels) - no need to memorise Important human and animal pathogens   ANAEROBIC CULTURE METHODS   **Anaerobic Chamber**   A chamber that doesn\'t allow oxygen inside. Because even a small amount of oxygen could kill the clostridia.   --- ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------    --- ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------   CLOSTRIDIAL ENDOPORES   *Clostridium* form **endospores** under adverse environmental conditions, such as the presence of oxygen **Spores** are a survival mechanism; is [insensitive] to oxygen E.g. Think like a seed - contains all the genetic material to grow again; has a hard outside shell and when the conditions are right, it can grow, otherwise is able to survive for a long time. Spores are characterised on the basis of position, size and shape, can be used for identification   Clostridium tetani Clostridium Clostridium cadaveris Clostridium bifermentans   SPORULATION      No need to memorise all the stages.   Very energy dense process.   BACTERIAL ENDOSPORE STRUCTURE       C. DIFFICILE SPORES HAVE AN EXOSPORIUM        Protein structures have a lot of carbohydrates on them and are really sticky which cause the spores to stick together and help them to stick to other surfaces to (e.g. Hospital surfaces)   THE PATHOGENIC CLOSTRIDIA   Neurotoxic C. tetani C botulinum Enterotoxic C perfringens C difficile Histotoxic C. perfringens tetanus botulism food poisoning, enteritis necroticans pseudomembranous colitis gas gangrene   **Common feature: produce potent protein toxins**   If they lose their ability to produce these toxins, they lose their pathogenicity.        **NEUROTOXIC CLOSTRIDIA**   **Clostridium tetani** -- causative agent of tetanus -- produces tetanus toxin neurotoxin -- pathogenesis entry of spore into deep wound (deep wound - cuts off blood circulation and therefore oxygen circulation, creating an anaerobic environment) germination of spore in anaerobic environment production of toxin by growing bacteria which is released when they die toxin binds to nerve endings and is translocated into nerve cells inhibits neurotransmitter release blockage of muscle relaxation pathway   PATHOGENESIS OF TETANUS   Results in uncontrolled stimulation of muscles -- tension, cramping twisting of muscles -- spasms and convulsions -- rigid paralysis -- death from spasms of the diaphragm and respiratory muscles   Vaccination using tetanus toxoid is an effective preventative measure   RIGID PARALYSIS   A naked person lying on the ground Description automatically generated   Sir Charles Bell: Portrait of a soldier with generalised tetanus Royal College of Surgeons, Edinburgh. Death follows bodily exhaustion and occurs by respiratory failure or circulatory collapse   **Clostridium botulinum** -- causative agent of botulism food-borne disease commonly from contaminated canned foods that have been inadequately heated (heating up something drives the oxygen out of the jar) -- pathogenesis spores germinate in food and toxin is produced during vegetative growth pre-formed toxin (BoNT) is ingested with food toxin localises in neuromuscular junction blocks release of neurotransmitter   PATHOGENSIS OF BOTULISM   Results in an uncontrolled relaxation of muscles -- symptoms within 18 to 24 hrs of ingesting toxin -- blurred vision, difficulty swallowing and speaking, muscle weakness, nausea, vomiting -- flaccid paralysis -- death due to cardiac or respiratory failure   No vaccine available -- can treat with antitoxin if administered quickly   THERAPEUTIC USES OF BOTULINUM NEUROTOXINS (BoNT)   Injection of low levels of BoNT into striated muscle leads to a reversable denervation of neuromuscular junctions. No effect on anatomical contact between nerves and muscle, no loss of motor axons. BoNT used to treat severe focal dystonias -- spasms and facial tics. BoNT used in cosmetic industry -- removal of wrinkles and decreasing perspiration Very safe, effects last 1 to 4 months   TETANUS VERSUS BOTULNUM TOXIN   --- ----------------------------------------------------------------------------------    --- ----------------------------------------------------------------------------------   **ENTEROTOXIC CLOSTRIDIA**   ENTEROTOXIC CLOSTRIDIA - HUMAN DISEASE   **Clostridium perfringens** -- aerotolerant anaerobe (wont grow in the presence of oxygen but may not die in the presence of small amounts of oxygen); forms spores -- can cause food poisoning present in meat that has been heated and cooled slowly ingested in huge numbers -- produces an enterotoxin bacterial cells sporulate in the intestine production of enterotoxin is associated with sporulation -- onset of symptoms after 8 to 16 hours watery diarrhoea, nausea, abdominal cramps   (third most common cause of food poisoning)   ORDER OF DISEASE EVENTS   strain colonisation; enteric toxin production; gut permeabilisation; dissemination of toxins outside the gut; toxins act on distant organs.   C. PERFINGENS TOXINS AND DISEASE   Enteric pathogen of humans and animals -- humans: food poisoning, gas gangrene -- animals: enterotoxaemic diseases Virulence largely attributed to the production of up to \>22 toxins, particularly in animal diseases However, not all isolates express all toxins Vaccines are often toxin-based   ANIMAL INFECTIONS   C. perfringens causes disease in many animals   Type A: lamb enterotoxaemia, avian necrotic enteritis, myonecrosis, equine colitis. Type B: lamb dysentery, ovine enterotoxaemia Type C: ovine, bovine, porcine enterotoxaemia, enteritis in foals Type D: ovine (pulpy kidney disease), caprine and bovine enterotoxaemia Type E: rabbit enteritis, rare cause of enterotoxaemia in other species.   Different types cause different diseases because they have different toxins.   Predisposing factors are often critical for susceptibility to these and many other clostridial enteric diseases.   Don\'t need to memorise \^\^   C PERFINGENS TYPE D INFECTIONS   A red line with black and white lines Description automatically generated   All types make the alpha toxin.   **Type D** strains colonise the gut. They won\'t cause disease without the predisposing factors: 1 - Carbohydrates in diet -- sudden changes to the diet 2 - Bacterial contamination levels (if a lot is being shed into the environment and the animals are ingesting it) 3 - Immune status - vaccination   PATHOGENESIS OF TYPE D INFECTIONS   1. **Pro-ε-toxin**: produced in intestine 2. activated by trypsin/other proteases (clip off the part that is holding the toxin in inactivity) 3. increases intestinal permeability (gut becomes leaky, things in the gut can leak into other tissues) 4. absorbed systemic circulation 5. endothelial cells of brain, kidney etc.   Can also get: 1. Pulmonary edema (fluid accumulation/swelling) 2. Pulpy kidney disease 3. Brain lesions in cattle   **ε-toxin is essential for type D disease**      When the epsilon toxin was knocked out showed us that the toxin has no impact. Therefore the epsilon toxin gene is responsible and important for these diseases   C. PERFRINGENS ENTERIC INFECTIONS   Strain colonises Produces enteric toxin Enteric toxin permeabilises the gut Toxins and other factors are absorbed via the 'leaky' gut Toxins act on distant organs   **HISTOTOXIC CLOSTRIDIA**   HISTOTOXIC CLOSTRIDIA - HUMAN DISEASE   **C. perfringens** -- causes **gas gangrene** (clostridial myonecrosis) injured tissue becomes contaminated with spores if tissue is anaerobic spores germinate and bacteria rapidly grow (e.g. 8/9 mins) extensive bacterial growth is observed   This can also be a problem for people with diabetes due to lack of circulation in extremities such as toes creating an anaerobic environment.   -- produces a-toxin phospholipase disrupts host-cell plasma membranes extensive destruction of cells and tissues   -- symptoms include severe pain, edema, muscle necrosis Characteristic lesions and blackening of the skin associated with gas gangrene are seen Treatment is radical and usually involves amputation of the affected tissue/limb   NORMLA HOST MICROBIOTA   Internal tissues usually free of microorganisms.   Surface tissues (skin, mucous membranes) in contact with environment, therefore colonised by bacteria, yeasts, fungi.   **Mutualistic relationship** - both host and microbiota derive benefit.   THE HUMAN GASTROINTESTINAL TRACT - NORMAL MICROBIOTA Up to 2x more bacterial cells than human cells. Weigh 2-3 kg, 1.5 L 300- 1000 different species Aquired after birth Change with age/diet/health First line Of defense against pathogens by occupying the niche: \"competitive exclusi90\'     NORMAL HOST MICROBIOTA   **Microbiota derives:** 1\. supply of nutrients 2\. stable environment (e.g. Stomach will be acidic..) 3\. constant temperature 4\. protection 5\. transport   **Host derives:** 1\. some nutritional benefit 2\. prevention of colonisation of pathogens   Some microbiota benefit from relationship, do not provide benefit to host, do not harm host: **commensal** relationship. If microorganism benefits at the expense of host, **parasitic** relationship. Disease-causing microorganisms are parasites.   NON-SPECIFIC IMMUNITY BY NORMAL MICROBIOTA   Bacterial antagonism by normal microbiota: Approximately 100 trillion bacteria and other microorganisms reside in or on the human body. These keep potentially harmful opportunistic pathogens in check and inhibit the colonisation of pathogens by:   1\. Producing metabolic products: Fatty acids, bacteriocins, etc. inhibit the growth of many pathogens. 2\. Adhering to target host cells: Prevent pathogens from colonising. 3\. Depleting nutrients essential for the growth of pathogens. 4\. Stimulating the immune system so it is primed to fight pathogens.   ANTIBIOTICS AND RESIDENT MICROBIOTA   Destruction of normal microbiota through the use of antibiotics (dysbiosis) leaves the host vulnerable to infections by opportunistic pathogens.   Dysbiosis caused by antibiotics pave the way for opportunistic pathogens:      NON-SPECIFIC IMMUNITY BY NORMAL MICROBIOTA   Destruction of normal microbiota by use of antibiotics leaves host vulnerable to infections by opportunistic microbiota.   Examples: Candida and Clostridium difficile. \- Held in check by normal microbiota. \- Not killed by antibiotics (why?) \* Candida causes thrush. \* C. difficile overgrows intestinal tract, produces toxin - antibiotic-associated colitis.   ENTEROTOXIC CLOSTRIDIA   **Clostridium difficile**: an opportunistic pathogen Leading cause of infectious diarrhoea in hospitals worldwide. Also a problem in the community and animals (pigs, cattle, horses) Strict anaerobe and spore former -- spore are critical for infection; their persistence is a problem. *C. difficile* only colonises gut if normal microbiota is disrupted -- usually via antibiotics.   Note Clostridium difficile renamed Clostridioides difficile recently   ANTIBIOTICS AND C. DIFFICILE INFECTION     1\. Patient resistant to CDI (c. Difficile infection) if normal microbiota not disrupted by antibiotics 2\. When antibiotic treatment commences, infection with resistant strain more likely while antibiotic is being administered 3\. When antibiotic treatment ceases, microbiota remains disturbed during which patients can be infected with resistant or susceptible C. difficile 4\. After microbiota recovers, colonisation resistance to C. difficile is restored.   C. difficile is a problem of antibiotic use.   THE C. DIFFICILE INFECTIOUS CYCLE   --- ------------------------    --- ------------------------   **C. difficile-mediated pseudomembranous colitis**   Gross section of the lumen of the colon showing antibiotic-associated colitis. Yellowish plaques of fibrin, mucus and inflammatory cells overlay the normal intestinal mucosa. Disease pathology results from toxin production.   MAJOR VIRULENCE FACTORS OF C. DIFFICILE: **TOXINS A AND B**   Located on a 19.6 kb pathogenicity locus (PaLoc) \- found in all toxigenic strains, absent in avirulent strains. Members of large clostridial glucosylating toxins family -- monoglucosyltransferases that glucosylate Rho GTPases. Toxin action results in severe damage to intestinal epithelium.     **WHAT DOES C DIFFICILE INFECTION AND THE TOXINS DO TO THE INTESTINAL EPITHELEIUM?**   INTESTINAL INTEGRITY AND MAINTENANCE   The integrity of the intestinal epithelium relies on -- Cellular polarity -- Formation and maintenance of tight junctions -- Renewal of the stem cell population and maintenance of the stem cell niche        **CELLULAR POLARITY AND INTESTINAL INTEGRITY**   Intestinal epithelial cells are prototypical polarised cells -- Distinct lumen-facing apices with microvilli -- Bases anchored to basal lamina   Cellular polarity is important for intestinal homeostasis If this is disrupted, the gut will not be able to function properly **Ezrin** is important for apical integrity in intestinal epithelial cells -- Can be used as a marker for cellular polarity   Examined using immunofluorescent staining and confocal microscopy      **CDI damages cell polarity and the colonic surface**       **Adherens junctions and intestinal integrity**   **Adherens junctions (AJ)** maintain intestinal integrity Connect actin cytoskeleton between cells Disruption of AJ can exacerbate colonic inflammation -- 'leaky' gut Used E-cadherin and βcatenin to assess AJ formation and integrity   Examined using immunofluorescent staining and confocal microscopy    \*White in the middle is space b/w cells   **C. difficile infection disrupts the junctions between cells**     Orange staining due to the mix of the green and red stain   **Intestinal integrity and maintenance**   The gut is the most rapidly proliferating tissue in adult mammals and the intestinal epithelium is constantly replaced The stem cells are responsible for repairing the damaged tissue -- **Stem cells** at the base of the epithelium give rise to new cells -- Migrate to crypt surface and are sloughed off through apoptotic death -- How is this process affected during C. difficile infection?      C. DIFFICILE INFECTION DAMAGES COLONIC STEM CELLS   Organoid culturing was used to determine that C. difficile infection affects stem cell function and gut repair capacity **Organoids** are 3-dimensional organ-bud grown in vitro from stem cells, that forms a 'mini gut'   A collage of images of different types of organ organs Description automatically generated   CDI ALTERS STEM CELL FUNCTION AND ORGANOID FORMATION   Organoid culturing was used to determine if C. difficile infection affects colonic stem cell function and gut repair capacity \- infection reduces stem cell function ex vivo      DOES TCDS AND TCDB ALTER HUMAN OGANOID FUNCTION?   How do **TcdA and TcdB** affect human organoids? -- Healthy tissue colonic organoids were cultured -- Subjected them to increasing purified toxin   A black and white image of a person Description automatically generated      Toxin b completely kills cells/ has the most dramatic effect   SUMMARY   Colonic structure is disrupted Integrity mechanisms are perturbed Adherens-junctions (cell-to-cell contacts) Ezrin (cell polarity) Stem cells and niche are damaged; repair capacity disrupted These effects result in severe damage and disease. Ø Other consequences?   C. DIFFICILE INFECTION AND SYSTEMIC DISEASE   C. difficile infection damages junctions between cells leads to 'leaky gut' Spores, toxins and other gut contents disseminate beyond the gut during severe disease. This occurs in many gut infections and other gut diseases e.g. irritable bowel syndrome (IBS) and inflammatory bowl disease (IBD) (ulcerative colitis, Crohn's disease).   CURRENY C. DIFFICILE TREATMENTS   Discontinuation of antibiotic (that induced susceptibility to the disease) and fluid replacement More antibiotics! -- Oral metronidazole (causes Side effects) and/or vancomycin Relapses occur because the microbiome keeps being disrupted by the antibiotics Other antibiotics are also undergoing trials or have been approved, (eg fidaxomicin; narrow host range - doesnt kill as much of the microbiome)   NEW C. DIFFICILE TREATMENTS UNDER CONSIDERATIONS   Non-antibiotic treatments have also been tested: Probiotics Intravenous IgG antibodies (containing human anti-toxin IgG) Monoclonal antibodies for passive immunotherapy, expensive and is not oral Passive polyclonal immunotherapy (cow colostrum - c. Difficile is injected into cow \--\> cow makes antibodies in gut \--\> those are taken out and given to patient; egg yolk antibodies) Faecal transplant therapy - the poo sample is blended \--\> the blended is given to patient   MICRIOBIOTA CAN BE RESTORED     C. DIFFICILE IS AN URGENT HUMAN THREAT      TRAVEL DISSEMINATES C. DIFFICILE STRAINS     WHERE DO C. DIFFICILE STRAINS COME FROM?      C. DIFFICILE INFECTIONS OF ANIMALS   Under-recognised cause of disease in animals -- neonatal pigs, beef and dairy calves, horses and companion animals. No precise estimates of economic burden. Can be detected in meat and meat products -- suggests foodborne transmission to humans. Can also be detected in water runoff and compost -- suggests other transmission portals, can be transmitted via compost   As with many bacteria, it is likely that there is a close relationship between animal and human strains and transmission: a "one health" problem. **Week 12 \| HPAI H5N1** ZOONOTIC SPILLOVER: A SOURCE OF NEW VIRUS INFECTIONS IN HUMANS   **Sporadic spill over event**: when an animal virus gets into the human population - usually occurs through an intermediate host.   CHARACTERISTICS OF A PANDEMIC   Associated with introduction of virus into human circulation with no prexisting immunity Sudden onset, no real warning Rapid global spread, easy human to human transmission Get waves of infection, increasing virulence   Two recent examples: Influenza (2009) and SARS-COV2   INFLUENZA A VIRUSES: STRUCTURE AND REPLIACTION CYCLE      Envelope virus RNA negative sense Matrix protein - important for uncoating of the virus; starts pumping hydrogen ions which makes a low ph resulting in disassembly of proteins **HA (Hemagglutinin) and NA (Neuraminidase):** These are two important surface proteins of the virus. HA is responsible for binding to host cells, while NA is involved in the release of new virus particles.  **Attachment:** The virus attaches to the surface of a host cell through the HA protein.  **Endocytosis:** The virus is engulfed by the host cell through a process called endocytosis, forming a membrane-bound vesicle.  **Fusion and Uncoating:** The viral envelope fuses with the vesicle membrane, releasing the viral RNA into the host cell cytoplasm.  **Translation:** The viral RNA is translated into viral proteins by the host cell\'s ribosomes.  **Post-translational Processing:** The newly synthesized viral proteins undergo modifications, such as glycosylation and phosphorylation.  **Packaging:** The viral RNA and proteins are assembled into new virus particles.  **Release:** The newly formed virus particles are released from the host cell by budding, acquiring a new envelope from the host cell membrane.   WILD AQUATIC BIRDS ARE THE NATURAL VIRAL RESERVOIR FOR INFLUENZA BIRDS       More migratory birds that travel a lot   HA ATTACHMENT: A TRMER THAT RECOGNISES SIALC ACIDS      Alpha helix sits on the inside of the haemagglutinin. The haemagglutinin binds to the salic acid residues that are found on sugars on host proteins.   DISTINCT INFLUENZA HAS CAN DISTINGUISH BETWEEN DIFFERENT SIALI ACID LINKAGES   **Sialic acid** is a sugar molecule found on the surface of many cell types, including those in the respiratory tract, which is a primary target for influenza A virus infection. The linkage between sialic acid and the underlying sugar (galactose) plays a crucial role in determining the specificity of influenza A virus binding. α**2,3 Linkage:** This type of linkage is predominantly found on the surface of avian cells, such as those in the respiratory tract of birds. Many avian influenza A viruses preferentially bind to α2,3 sialic acid. α**2,6 Linkage:** This type of linkage is more prevalent on the surface of human cells, especially in the upper respiratory tract. Most human influenza A viruses preferentially bind to α2,6 sialic acid.   The sialic acid gets linked to the galactose in 2 ways as above via the two different linkages   Depending on how they're linked gives a different shape; meaning that different haemagglutinin will be restricted depending on whether or not they bind to the alpha 2,3 or alpha 2,6.   HAEMAGGLUTININ BINDS TO SIALIC ACID GROUPS ON CELL SURFACE PROTEINS      The type on linkage is relevant because it explains why bird viruses do not readily get into the human population because humans have an enrichment of alpha 2,6 and birds have 2,3.   CLEAVAGE OF HA BY HOST PROTEASES IS KEY FOR PENETRATION AND UNCOATING     **HA Cleavage:** **- Cleavage Site:** The HA protein has a specific cleavage site located between the HA1 and HA2 subunits. **- Host Proteases:** This cleavage site is recognized and cleaved by host proteases, such as trypsin-like proteases, which are found in the respiratory tract. **- Activation:** Cleavage of the HA protein is essential for its activation and the ability of the virus to infect host cells. The cleavage of the HA protein occurs in a low pH environment, such as within an endosome after the virus enters the host cell. This acidic environment is necessary for the proteases that cleave the HA protein to function. **Impact of Cleavage:** **- Penetration:** Cleaved HA is required for the virus to penetrate the host cell membrane. **- Uncoating:** Cleavage also facilitates the release of the viral genome into the host cell cytoplasm. In humans, the protease is only found in the lungs. This is why influenza cause respiratory infections.   The cleavage events forms 2 subunits: hA1 and hA2. As a result, the haemagglutinin is able to undergo conformational change which happens when there is a decreae in pH. The decrease in pH results in firing of the hairpin.    CHARACTERISTICS OF HUMAN FLU   Spread by aerosol and/or droplet Rapid spread of infection Several days before onset of symptoms Virus shed before onset of symptoms   INFLUENZA A VIRUS INFECTION INDUCES A ROBUST IMMUNE RESPONSE   Symptoms include fever, pneumonia (take a 5-7 days to appear); due to production of cytokines Generate excellent antibody response to spike (HA\> NA) proteins; provide protective immunity Generate excellent T cell responses (cell mediated immunity)   Individuals most at risk from Influenza A infection are: Young, elderly, immunocompromised   ADAPTIVE IMMUUNITY IS MEDIATED BY      INFLEUNZA A VIRUS IMFERCTION INDUCES A ROBUST IMMUNE RESPONSE     The antibodies bind to the virion and prevent attachment and therefore infection.   ANTIGENIC DRIFT      Because the virus is RNA, it doesn't have any proof reading mechanism. Which means that it is more likely to get mutations. These mutations could result in changes in the **spike proteins**. (spike proteins = HA and NA)   ACCUMULATIONS OF MUTATIONS IN ANTIBODY BINDING SITE OF HA   Therefore, immunity to previous year's strain not as effective   RAPID SPREAD OF SEASONAL INFLUENZA      Mismatch: the immunity induced by the vaccination does not match the virus strain for the season.   PROTECTION FROM INFLUENZA IS PRIMARILY VIA NEURALISING ANTIBODY   A diagram of a virus Description automatically generated Antibody-mediated immunity alone may not be sufficient to protect against all influenza A virus subtypes. This is due to the diversity of influenza A viruses and the ability of the virus to undergo antigenic drift, which involves small changes in the HA and NA proteins that can reduce the effectiveness of existing antibodies. ANTIGEN SHIFT CAN RESULT IN A NEW HUMAN SUBTYPE (PANDEMIC STRAIN)          New haemagglutinin + human virus = new virus A FLU PANDEMIC AFTER INTRODUCTION DIRECTLY FROM ANIMAL SPECIES (NO NEED FOR REASSORTMENT)   0 丨 apod   1981 SPANISH FLU      Due to the spanish flu   THE 1918 SPANISH INFLUENZA PANDEMIC - THE MOTHER OF ALL PANDEMICS   SAVE YOURSELF FOLLOW TWO SIMPLE RULES Wear a Mask Spmish Innu e n n Estimated that 30-50 Million died from Spanish influenza   This was a significant proportion of the population at the time   THE WAVES OF INFLIENZA OUTBREAKS   There were waves of influenza outbreaks during 1918/19.        Soldiers were going home after war: more spread \--\> spring wave   Large proportion of the population that were getting sick were healthy population.   RESURRECTING THE 1918 SPANISH FLU   〔 e i , n of the 1918 influenza virus polymerase genes al, N 2 05 , 43 889   He was able to resurrect the spanish flu jurassic park style. He got these from dead buried people in the ground in alaska, which meant the bodies were preserved ad frozen.   To do so, he did a PCR of the DNA sequence of each of the gene segments for the 1918 spanish flu. The gene sequences were cloned into plasmids. Reverse genetics: giving some cell things to the plasmid so that in can translate and transcribe to produce the spanish flu.   1918 SPANISH FLU DEMONSTRATED ADAPTATION TO THE HUMAN HOST    The first wave \--\> explains how the virus can transfer from birds to humans. Due to both the alpha linkages being attackable. (2,3 for birds and 2,6 for humans)   Bronchus \--\> predominant linkage is alpha 2,6 Bronchioles and alveoli \--\> predominant linkage is alpha 2,3   Essentially, if the bird virus can get far down enough, it can affect human tissue. However, like the first wave, if the virus can see both alpha 2,3 and alpha 2,6, it increases the likelihood of the virus attaching itself at more points of the respiratory tract.   The virus will want to replicate as high as it can in the airways so that when you cough or sneeze, it will be able to spread better.   RESURRECTED 1918 SPANISH INFLUENZA   flu flu Spanish flu causes prolonged inflammation in the infected lung   BIRD FLU (H5N1/H7N9) A PANDEMIC WAITING TO HAPPEN?   Two types of bird flu (domestic poultry):   **Low pathogenic avian influenza (LPAIs)** Minor or no symptoms   **Highly pathogenic avian influenza (HPAIs)** Evolve from LPAIs Systemic infection, hemorrhagic, very virulent Best way to get rid of it is to kill all the birds that have it and the birds that surround the birds with the virus.   HPAI OUTBREAKS    HPAI outbreaks used to be quite rare   USEFUL CHARACTERISTICS OF HPAI OUTBREAKS   Virulence associated with insertion of multiple basic amino acids in cleavage site of HA More efficient cleavage of HA by proteases such as plasmin and furin (systemic) This means that the virus will not remain localised to the upper respiratory tract Direct transmission from birds to human, no need for pig as intermediate host \- evidence this was also the case for Spanish influenza pandemic H5N1 viruses have become endemic in wild aquatic bird populations Continuing to evolve in bird reservoir in S.E Asia, Southern China H5N1 viruses have not yet acquired the ability to spread from human to human efficiently.   Spread in birds: A map of the world with different colored circles Description automatically generated   Spread in humans:    WHY CAN AVIAN H5N1 INFECT HUMANS BUT NOT TRANSMIT HUMAN TO HUMAN?   Basically we have more alpha 2,3 in the upper airways. And we need he alpha 2,6 linkage to be affected to be infected. Therefore it is difficult to cough up the 2,6 because it is so low in our respiratory tract making it difficult to spread. And because it is so low, it is difficult for the virus to reach that low in our respiratory tract.     H5N1 PATHOGENICITY IS MULTI-FACTORIAL AND CONSTANTLY EVOLVING   Ι 196 Ρ« 92 Ιη de (1 2.3GOl llokoge Ιη cell     HUMAN INFECTION IS INEVITABLE        WHY BE CONCERNED ABOUT H5N1?   Direct transmission from birds to human, no need for pig as intermediate host -evidence this was also the case for Spanish influenza pandemic H5N1 viruses have become endemic in bird populations Continuing to evolve in bird reservoir in S.E Asia, Southern China H5N1 viruses have not yet acquired the ability to spread from human to human efficiently   IS H5N1 ADAPTING TO BETTER INFECT HUMANS?   p!es suadxa \'MOI sugełuał O!łqnd atp Ol queaq aqł pue \'sueułnq auotue Kusea pea.lds 01 01 SIDUIWDW u0YDJdDPVJO sufis awos pamoqs uDW uoapqo zuoy ałdwos nu pąg   INFECTION OF MAMMALS BY H5N1 HPAI (USA)        MOST RECENT OUTBREAK OF H5N1 IN HUMANS   Poultry outbreaks On the last week) Non-poultry (in the last week) 1 Includes 2 human infections   EMERGENCE OF H5N1 HPAI IN USA DAIRY HERDS        BIRD FLU (H7N9) ANOTHER VIRUS TO WORRY ABOUT?   795 сазе, ЗИ аесн„ но„д co„ada, Mclaya,io)   HOST RANGE OF H5N1 IS GROWING: A SOURCE OF A NEW VIRUS INFECTIONS IN HUMANS