Virology Module 4 - PDF

Summary

This document is a module on virology, discussing origins, structure and replication, at a cellular level. It includes topics such as virus entry, viral protein synthesis, genome replication, and virus assembly with a focus on different types of viruses including naked and enveloped. It includes practice questions in the latter section.

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L11: Origin, Structures and Definition of Viruses Microbiology & Immunology Courses (MICR2000) Table of contents What is a virus? Ch...

L11: Origin, Structures and Definition of Viruses Microbiology & Immunology Courses (MICR2000) Table of contents What is a virus? Characteristics of a virus Composition of a virus The Nucleocapsid Naked and Enveloped viruses Enveloped virus structure Complex viruses Summary Practice questions What is a virus? A structure that has evolved to transfer nucleic acid (genetic element) from one cell to another genetic element that cannot replicate independently of a living (host) cell outside a living cell a virus is an inert macromolecule viruses are very small 20-350nm (mimivirus ~ 700nm) only poxviruses and mimiviruses are visible by light microscopy L11: Origin, Structures and Definition of Viruses 1 Bacteria > 1um Pandora virus (~1um) Characteristics of a virus Intracellular parasite Cannot replicate outside a living cell Large inert macromolecules Possess only one kind of nucleic acid DNA or RNA - not both Limited amount of genetic material 3 genes in simple viruses 200 genes in complex virus for context: bacteria > 500 genes Viruses - not enough genetic information to code for energy production of high potential; Do not possess ribosomes: Do not grow from the integrated sum of their constituents → they replicate; Viral nucleic acid and viral protein synthesis occur separately - come together during maturation RNA viruses are only organisms to use RNA as genetic material Lipid and carbohydrate are acquired from host cell (not coded for by viral genome) Composition of a virus All viruses contain a nucleic acid genome (RNA or DNA) and protein Protein usually coded for by viral genome some protein acquired from host during replication Structural proteins → part of virus particle (virion) L11: Origin, Structures and Definition of Viruses 2 cell attachment and penetration virus assembly and release protection of nucleic acid and (replication enzymes in some viruses) Non-structural proteins → most not found in virion replication (polymerases, helicases) virus assembly and release (proteases) The Nucleocapsid The viral genome is surrounded by a protein capsid composed of many subunits of one or more structural proteins capsomers The capsid together with the genome form the nucleocapsid Capsomers are arranged either in icosahedral or helical symmetry Icosahedral nucleocapsids Icosahedral virus the capsomers are arranged to form equilateral triangular faces may appear spherical Helical nucleocapsids Helical symmetry → capsomers are arranged in helical pattern around a central core Helical nucleocapsids tend to be rod-shaped rather than spherical L11: Origin, Structures and Definition of Viruses 3 Naked and Enveloped viruses Viruses composed only of nucleic acid and protein are called naked viruses often vary stable in the environment nucleic acid ‘encased’ in protein e.g. FMDV Viruses that acquire an outer layer of membrane from the host during replication are called enveloped viruses enveloped viruses generally more fragile more susceptible to inactivation because of lipid content (e.g. HIV) L11: Origin, Structures and Definition of Viruses 4 The envelope is acquired by the virus as it matures and buds through the cell membrane virus encoded proteins synthesised in infected cell and incorporated in cell membrane E.g. Spike proteins (peplomers) acquired by virus as it buds through host cell membranes Enveloped virus structure Enveloped viruses may have icosahedral or helical nucleocapsid all helical viruses of animals are enveloped naked helical viruses only found amongst plant and bacterial viruses L11: Origin, Structures and Definition of Viruses 5 Examples of enveloped viruses → helical nucleocapsids Orthomyxoviruses (e.g. influenza) Paramyxoviruses (e.g. measles, mumps, Hendra) Filoviruses (e.g. Marburg, Ebola) Helical nucleocapsids - flexible virion structure → can be spherical, pleomorphic, filamentous Examples of enveloped viruses → icosahedral nucleocapsids Togaviruses Ross River Rubella Flaviviruses Dengue Yellow fever Herpesviruses Herpes simplex Chicken pox Enveloped viruses vary in shape L11: Origin, Structures and Definition of Viruses 6 Complex viruses Neither helical nor icosahedral Large viruses with complex structure Poxviruses found in mulberry or ball of yarn shape T-even bacteriophage Icosahedral head Contractile tail/sheath Base plate Tail fibres/legs Mimivirus Giant DNA virus infecting species of genus Acanthamoeba Particle size of 0.7um, most complex virus L11: Origin, Structures and Definition of Viruses 7 Summary The structure of a virus is determined by: Side and coding capacity of the genome Genome ahs to fit into a nucleocapsid Number of structural proteins available Most economic nucleocapsid symmetry Functional requirements also important: Protection of genome from environment Mode of cell attachment and entry Mode of replication Mode of virion assembly and release Summary of Viral components Capsid: protein shell that encloses the nucleic acid Capsomers: individual subunits that make up capsid Nucleocapsid: the capsid and the enclosed nucleic acid Envelope: lipid membrane acquired by nucleocapsid as it buds from cells may contain proteins and sugars of host cell and viral origin The Virion is the complete infective virus particle (including envelope) Structural Analysis of viruses Electron microscopy (EM) Examination of virus structure at low resolution: virus identification, cellular location L11: Origin, Structures and Definition of Viruses 8 X-ray crystallography High resolution of structure of virions or viral protein structure: virus/receptor interactions identification of precise targets for antiviral agents Major differences between viruses and bacteria Practice questions 1. List the major difference between viruses and bacteria 2. How do viruses acquire an envelope? How are enveloped viruses different from naked viruses? Give examples of each! 3. What are the different structural components of a virus? L11: Origin, Structures and Definition of Viruses 9 4. List the two major symmetries that viruses utilise and give an example for each. 5. What factors determine the structure of a virus 6. Viruses are thought to have made important contributions to the evolution of cellular life – discuss this statement including theories on the origins of viruses L11: Origin, Structures and Definition of Viruses 10 L12: Replication of Viruses Microbiology & Immunology Courses (MICR2000) Table of contents Aims of the lecture: Attachment & Entry Adsorption (attachment) Penetration Entry of naked viruses Initiation of Replication (Uncoating) Synthesis of viral protein and nucleic acid Viral genomes DNA viral genome → mRNA RNA viral genome → mRNA Replication of + strand RNA virus Replication of - strand genome Viral maturation and release Release → naked and enveloped viruses Antivirals Targets for antiviral agents Antiviral drugs Practice Questions Aims of the lecture: To understand the basic stages of virus replication at the cellular level 1. Virus entry into susceptible cell 2. Viral protein synthesis 3. Viral genome replication 4. Virus assembly 5. Release of mature virion from cell L12: Replication of Viruses 1 Attachment & Entry Early phases of replication for animal viruses: adsorption (attachment) penetration uncoating Adsorption (attachment) Brownian motion allows random collisions of virus with cell receptor Virus attachment sites: Enveloped viruses - spike/peplomers Naked virus anywhere on the virus surface (capsid) Cell receptors cell surface molecules e.g. CD4 and CCR5 for HIV Attachment physical complementarity between virus and receptor specific interaction - most viruses can only infect some cell types results in host and tissue specificity L12: Replication of Viruses 2 Penetration Viruses enter cells by various modes: Endocytosis - virus is engulfed into cytoplasmic vacuole Membrane fusion (enveloped viruses) Viral envelope fuses with cytoplasmic membrane Only the nucleocapsid enters the cell Direct entry (some naked viruses) The virus shell (capsid) undergoes molecular rearrangement Whole virus or viral genome enters L12: Replication of Viruses 3 Entry of naked viruses Direct entry The nucleocapsid undergoes molecular rearrangement allowing viral genome to enter the cell Exosomes Naked viruses are excreted from infected cells in exosomes and enter new cells in membrane bound particles Initiation of Replication (Uncoating) Uncoating allows release of the genome into the cytoplasm Often spontaneous also carried out by host cell or viral enzymes - protease, lipases etc.) Some viruses shut down host cell macromolecular synthesis early in replication Viruses don’t like competition The cell is instructed to synthesize only viral proteins Some enveloped viruses require healthy host cells to actively synthesize cellular proteins and membranes for virus replication Synthesis of viral protein and nucleic acid DNA viruses replicate in the cell nucleus All DNA replicative enzymes are found in the nucleus Except poxviruses L12: Replication of Viruses 4 RNA viruses generally replicate in the cytoplasm Some exceptions → e.g. retroviruses, influenza HOST RNA is not replicated in cells RNA viruses code for replicative enzymes (proteins) but need polyribosomes to translate them from mRNA Many enzymes needed for viral replication are not coded for by the virus The virus uses host cell enzymes Viral genomes L12: Replication of Viruses 5 DNA viral genome → mRNA Double stranded DNA Single stranded DNA (+) Enter cell Synthesis of other strand L12: Replication of Viruses 6 Transcription of the minus ds DNA intermediate (-) strand Transcription of minus (-) Serves as a template for strand mRNA (+) Serves as a template for mRNA encodes proteins mRNA (+) mRNA encodes proteins RNA viral genome → mRNA Double stranded RNA virus (+) Make a (+) copy of the (-) strand RNA mRNA is made from there Single stranded RNA virus (+) Genome used directly as mRNA (+) Single stranded RNA virus (-) Make a (+) of the (-) strand RNA mRNA is made from there Single stranded RNA retrovirus (+) Reverse transcriptase drives reverse transcription of the RNA into a DNA intermediate (double stranded) DNA intermediate is used for transcription of the minor strand for (+) RNA synthesis Replication of + strand RNA virus L12: Replication of Viruses 7 Replication of - strand genome Virus enters cell Negative strand genomic RNA released into cytoplasm The replicase enzymes are brought into the cell as part of the virion Negative (-) strand RNA must first serves as template to make complementary positive (+) strand RNA for translation of viral proteins L12: Replication of Viruses 8 Viral maturation and release L12: Replication of Viruses 9 After viral nucleic acid and protein synthesis is complete → assembly occurs Assembly relatively spontaneous for simple viruses (self-assembly) More complex viruses may require various stages of assembly The strategies used by naked and enveloped viruses differ significantly Release → naked and enveloped viruses Naked viruses accumulate in cell Cell bursts open to release progeny viruses Release of progeny virus is sudden and complete (Virus may also be secreted gradually from intact cell in exosomes) Enveloped viruses bud through cell membrane released gradually L12: Replication of Viruses 10 Antivirals Targets for antiviral agents Viruses use host cell machinery for: Protein synthesis host translation system host enzymes for some protein processing Cellular enzymes for DNA replication (DNA viruses) Cellular energy to drive synthesis and enzyme reactions Attempts to interfere with replication by anti-viral drugs may also be toxic to cells Anti-viral agents usually target viral-specific enzymes or nucleic acid Antiviral drugs Acyclovir → e.g. Herpes simplex virus (HSV) Why not toxic to host cells? Drug only in active form in infected cells (activated by a viral enzyme that is only produced in infected cells Practice Questions 1. Briefly describe the replication strategy of : a) A positive strand RNA virus b) A negative strand RNA viruses Give two examples of each 2. The genomes of most DNA viruses replicate in the cytoplasm while most RNA viruses replicate in the nucleus. True or False? 3. List six (6) different types of viral genomes (eg. Double stranded DNA) L12: Replication of Viruses 11 4. List the basic stages of virus infection of the host cell (ie, attachment etc) 5. Research two types of antiviral drugs (ie. list two different modes of action) that are used to treat influenza infections - for each give an example of a drug (chemical or trade name) L12: Replication of Viruses 12 L13: Pathogenesis and Transmission Microbiology & Immunology Courses (MICR2000) Table of contents Lecture objectives: Evolutionary conflict → viral pathogenesis Viral disease syndromes Host factors modifying viral pathogenesis Routes of entry Skin Genitourinary Tract Respiratory tract Gastrointestinal tract Localization and Spread Modes of viral transmission Acute, Chronic, and Recurrent Infections Practice Questions Lecture objectives: Know the different types of viral infections and the disease syndromes they cause in animals Learn how virus: Enters and spreads within the body Where it replicates - how it is shed How it is transmitted L13: Pathogenesis and Transmission 1 Understand the events in viral infection that cause disease symptoms Evolutionary conflict → viral pathogenesis Both the host and virus are seeking a reproductive advantage New viruses are evolving continuously Natural selection favours viruses with low pathogenicity/virulence (so they don’t eradicate their hosts) most viral infections are asymptomatic disease is an ‘unusual’ consequence of infection The majority of COVID-19 cases are mild or asymptomatic L13: Pathogenesis and Transmission 2 Viral disease syndromes Fever Respiratory Disease Rash/Lesion Disease of CNS Hepatitis Oncogenic Disease Haemorrhagic fever Immunodeficiency Gastroenteritis L13: Pathogenesis and Transmission 3 What causes symptoms? 1. Damage to cells due to virus replication: cell death by rupture of cell during virus release (necrosis) cell commits suicide in response to infection (apoptosis) infected cell loses function → e.g. cytokine production infected cell transformed by virus → tumours HBV infection ⇒ chronically infected hepatocyte (liver cell) → HBV replication and insertion of virus genes into cell genome → activation of oncogene ⇒ malignantly transformed hepatocyte (cancerous liver cell) OR L13: Pathogenesis and Transmission 4 → HBV replication → Trans activation of oncogene by HBV X-gene → cancerous cell OR → Chronic tissue damage → Faulty DNA repair → cancerous cell 2. Damage due to the host response to infection: Immunopathology - antibodies and immune cells destroy infected cells → tissue damage Fever - elevated temperature stimulates immune response inhibits viral replication (most viruses temp. sensitive) Inflammation - symptoms of immune cells infiltrating area where virus infection has occurred (swelling, rash, pneumonia, etc.) COVID-19 Pathogenesis L13: Pathogenesis and Transmission 5 Host factors modifying viral pathogenesis Age Some viral infections more severe in different age groups: Covid-19, measles, chickenpox, Ross river and dengue relatively mild in children COVID-19, West Nile and flu usually worse in elderly Immune system maturity/waning → doesn’t respond to pathogens in the same way Hormonal influences Genetics e.g. HIV - cellular receptors are CD4 and CCR5 people with two copies of a mutant, shortened form of CCR5 have reduced susceptibility to HIV-1 infection L13: Pathogenesis and Transmission 6 but people with CCR5 mutation more susceptible to West Nile disease Metabolic state Generalised malnutrition or Vitamin D deficiency increase susceptibility to, and severity of measles infection Pregnancy (with its associated change in hormonal balance) can also lead to altered susceptibility to certain viruses Altered immune responses Impaired 1. genetically determined, e.g. agammaglobulinemia 2. acquired as a consequence of infection, e.g. HIV → weakens immune system (people can die from secondary infections) 3. iatrogenically (therapeutically) acquired, e.g. after transplant Enhanced auto-immunity Routes of entry skin respiratory tract gastrointestinal tract genitourinary tract conjunctiva Skin L13: Pathogenesis and Transmission 7 May be penetrated by viruses as a result of mechanical trauma (HPV, HIV, HSV, HBV, poxvirus) by injection (HBV, HIV) by the bite of an infected mosquito (arboviruses) either mechanical or true insect- borne by the bite of an infected animal (rabies) Generally viruses do not multiply locally but are carried away from site of infection by: bloodstream (HBV, arboviruses) migration along nerves (rabies) Genitourinary Tract Tears or abrasions allow viral entry Sexually transmitted viruses HIV herpes simplex (mostly HSV II) papilloma viruses (genital warts) hepatitis B virus Nature of cervical mucus, the pH of vaginal secretions and the chemical composition of urine all play a role in host defence L13: Pathogenesis and Transmission 8 Respiratory tract Major route of invasion: for viruses causing local respiratory infections SARS-CoV-2, flu, RSV, rhinoviruses others causing asymtomatic initial infection then generalised spread measles, mumps, chickenpox transmission usually by droplet infection in aerosols Gastrointestinal tract May involve; local infection (rotavirus, norovirus, adenovirus) or invasion of the host to produce systemic illness (polio, hepatitis A) due to invasion of tissues underlying the mucosal layer Virus survival depends on: acid stability resistance to bile salts inactivation by proteolytic enzymes Most-nonenveloped Localization and Spread L13: Pathogenesis and Transmission 9 Localization versus systemic spread many viruses multiply in epithelial cells at site of entry produce a spreading infection then shed directly to exterior respiratory infections - SARS-CoV-2, influenza, rhinoviruses and RSV gastrointestinal infections caused by rotaviruses dermatologic infections of the papillomaviruses polarized infection of epithelial cells and spread targeting of viral budding to apical or basal surfaces of polarized cells may define subsequent spread by viral glycoproteins → secreted proteins carry signals for targeting L13: Pathogenesis and Transmission 10 Modes of viral transmission L13: Pathogenesis and Transmission 11 Transmissibility/contagiousness of viral diseases L13: Pathogenesis and Transmission 12 Acute, Chronic, and Recurrent Infections Acute: rapid development of symptoms usually complete recovery or death e.g. yellow fever, flu, colds Chronic or persistent: long, slow infections (HIV, HCV) may have insidious onset (no symptoms) symptoms may be present most of the time sometimes continued symptoms for life sometimes complete recovery Recurrent or Latent: re-occurrence of symptoms from virus which has been latently present e.g. (herpes simplex) L13: Pathogenesis and Transmission 13 L13: Pathogenesis and Transmission 14 L13: Pathogenesis and Transmission 15 Practice Questions What events in viral infection can cause disease symptoms? Give examples! How do host factors modify the outcome of viral pathogenesis? Give examples! Describe the different types of viral infection in terms of the timing and appearance of disease symptoms and clearance of the virus from the body – give examples? L13: Pathogenesis and Transmission 16 Different viruses may infect similar epithelial cell surfaces, however their pathogenesis, in terms of the organs/tissues in the host that are affected, may vary greatly. Explain with examples! L13: Pathogenesis and Transmission 17 L14: Viruses of Plants and Bacteria Microbiology & Immunology Courses (MICR2000) Table of contents Plant Viruses Structure Types of plant viruses Plant virus transmission Classification and names Virus in a fungus in a plant Bacterial viruses Complex Phage Attachment Virulent Phage (e.g. T4) Temperate Phage Reproduction of temperate phage Gene transfer → transduction Bacteria vs phage Uses of plant viruses and phage in biotech Use of phage as medical antimicrobials Viruses of Archaea Practice Questions Plant Viruses Cause of many agricultural diseases banana brunchy top L14: Viruses of Plants and Bacteria 1 Fiji disease, sugar cane mosaic tobacco mosaic (Tobacco, tomato) barley yellow dwarf virus Structure Icosahedral or helical symmetry Naked (most) or enveloped (few) Most plant viruses have positive strand RNA genomes small genomes are better for cell to cell spread in plants Some have DNA genomes dsDNA circular genomes ssDNA circular genomes Multipartite Plant Viruses Viruses with segmented genomes with different genomic sections in different virus particles Plant cell must be infected with viruses containing each of the genomic sections for productive infection and replication In contrast most animal viruses with segmented gneomes contain all all genomic segments in a single virus particle (e.g. influenza) L14: Viruses of Plants and Bacteria 2 Types of plant viruses L14: Viruses of Plants and Bacteria 3 Tobacco Mosaic virus (TMV) First discovered virus in the 1890’s Positive strand RNA virus Naked helical virus Infect tobacco and tomato plants mottle appearance localised leaf lesions Plant virus transmission Plants have a rigid cell wall Cannot enter cells by endocytosis or fusion Enter by mechanical means Vector (common) Shearing, cutting (herbivores, contaminated tools etc.) Infected seeds Vectors Nematode worms Insects Aphids Leaf hoppers, white flies, mealybugs, thrips, mites and beetles L14: Viruses of Plants and Bacteria 4 Systemic infection and cell-to-cell spread After vector introduces virus: systemic infection via phloem Virus must bypass rigid cell wall barrier to spread from cell to cell Thin trans-wall channels that connect cells - plasmodesmata Plant viruses code for movement protein - facilitates movement of virus through plasmodesmata Mechanisms of cell-to-cell movement Encode for movement proteins which for tubules that insert themselves into the plasmodesmata, allowing the virus to travel through and move from cell to cell Encode for movement proteins that wedge open the plasmodesmata at the opening enough for viral particles to move through Classification and names Many virus families include both plant and animal viruses L14: Viruses of Plants and Bacteria 5 e.g. Rhabdoviridae, Reoviridae, Bunyaviridae Some virus families include only plant viruses e.g. Tobamoviridae, Geminiviridae, Potyviridae Most plant viruses nameed after disease symptoms in host e.g. banana bunchy top virus, broad bean wilt virus, barley yellow dwarf virus, tobacco mosaic virus Virus in a fungus in a plant Viruses and fungi can help wild plants adapt to extreme conditions In plants that thrive in hot, goethermal ground in Yellowstone National Park, viruses and fungi work together with plants to confer temperature hardiness Fungi and a type of grass grow together in tempeartures above 52 degrees celsius. If the plant and fungus are separated, however, both die in the same heat levels The fungus needs to be infected with the virus Bacterial viruses Known as bacteriophage or phage Structure: Most phage are naked Usually only protein and nucleic acid Genomes are mostly dsDNA some are ssDNA, ssRNA, or dsRNA Simple (icosahedral or helical) OR complex Complex Phage L14: Viruses of Plants and Bacteria 6 Most complex and largest: T even phages (e.g. T4, T2) Icosahedral head Collar or neck Helical sheath Base plate (Endplate) Tail pins and fibres Large dsDNA genome Linear or circular Attachment Brownian motion of phage particles results in random collisions - occasional attachment Complex phage - attach via tail fibres/base plate Simple phage - attach via capsid Some phage attach: 1. directly to cell wall 2. via sex pili Virulent Phage (e.g. T4) L14: Viruses of Plants and Bacteria 7 Virulent phages reproduce by Lytic cycle results in death of host bacterial cell 1. T4 Phage attaches to host cell by receptors for tail fibers 2. The sheath compresses, making a chole in the membrane 3. Genome is injected through the hole 4. Phage proteins and DNA are made 5. New phage particles assembled 6. Phage produces lysozyme which begins to digest the cellw all 7. Osmotic pressure causes the cell to burst and release phage particles Temperate Phage Lytic vs Lysogenic cycles L14: Viruses of Plants and Bacteria 8 Reproduction of temperate phage Some prophage (host cell genome with integrated viral DNA) genes are expressed Phage repressor protein prevents phage entering virulent replication cycle immunity of cell to further phage infection Expression of prophage genes may also affect virulence of bacteria e.g. Diptheria, botulism and scarlet fever are caused by bacteria carrying prophage genes that allow the bacteria to make toxins L14: Viruses of Plants and Bacteria 9 Gene transfer → transduction Bacteria vs phage Protection of bacteria from virulent phage: Many bacteria have restriction endonucleases restriction enzymes that cut up foreign (phage) DNA host cell’s DNA is modified so is resistant to restriction enzymes T4 survival strategy T4 contains a unique base that preotects it from REs L14: Viruses of Plants and Bacteria 10 Uses of plant viruses and phage in biotech Plant virusescode for foreign genes: espress foreign protein in infected plant tissue e.g. insecticide (resistance to pests) e.g. immunogenic protein Phage display - phage engineered to display foreign protein on their surface - useful expression vector e.g. production of synthetic antibodies Phage as tools in health and hygiene: Phage typing of bacterial pathogens highly host specific Use as antibacterial agents As preservatives The FDA have approved the use of phage as food additive - sprayed on surface of processed meat and poultry products to kill Listeria bacteria L14: Viruses of Plants and Bacteria 11 Bacteriophages in PhagoBioDerm help clear a wound from multidrug resistant S. aureus Phages can be administered in various ways orally, rectally, locally (e.g. skin), aerosol, and intravenously Use of phage as medical antimicrobials Advantages Disadvantages - Phages are very specific - do not harm humans or useful - No/few internationally recognised studies that bacteria → no side effects like prove the efficacy of phages in humans. bacteria - Multiply at site of infection until - Specificity can be a disadvantage when the exact there ar eno more bacteria species of infecting bacteria is unknown - Evolve and are found throughout nature → easy to find - Bacteria can become resistant to phages new phages - Phages are active against Phages are relatively large → cannot access some bacteria that have become sites in the body (inside cells etc.) antibiotic resistant - Phages can be genetically - When used intravenously - antibodies against modified in order to make up for phage produced → may be single use some disadvantages - Phages may transfer toxin genes between bacteria - Phages are more difficult to administer than antibiotics Viruses of Archaea L14: Viruses of Plants and Bacteria 12 Practice Questions 1. Briefly describe the lytic and lysogenic cycles of bacteriophage 2. What are prophage? How can they affect their bacterial host? 3. Do bacteriophage have any medical significance? Explain! 4. Some plant viruses have multipartite genomes. Briefly explain! 5. How do viruses infect plants and spread from cell to cell and establish systemic infection within the plant? 6. How can bacteriophage and plant viruses be useful? L14: Viruses of Plants and Bacteria 13 L15: Vaccines Microbiology & Immunology Courses (MICR2000) Table of contents Learning Objectives What is a vaccine? Adaptive Immune response to infection Types of Vaccines Adjuvants (non-living viruses) Living vs. Non-living vaccines Live Attenuated Vaccines Vaccine safety Naked DNA Vaccines Concerns with Naked DNA Vaccines Use of Vaccines SARS-CoV-2: Vaccines mRNA vaccines Needle free vaccine: Vaxxas High Density Microarray patch (HD-MAP) Virus neutralisation Protection from lethal SARS-CoV-2 challenge Practice Questions Learning Objectives What is a vaccine? Why use vaccine? How do vaccines work? Types of vaccines? What is a vaccine? L15: Vaccines 1 A vaccine is a preparation derived from a pathogen that when administered to the host does not cause disease but induces protective immunity against the pathogen The vaccine aims to prime the adaptive immune response to the antigens of the pathogen so that a first infection induces a secondary immune response The ideal vaccine should be effective, safe, stable and cheap Eradication of human diseases using vaccination: Smallpox Polio and measles (almost) Vaccination protects the vacinee and maintains herd immunity Controls transmission and spread Polio eradication WHO and Rotary launched polio eradication program in 1988 Resulted in a 99% reduction in polio cases Type 2 polio - officially eradicated Type 3 polio - oficially eradicated Adaptive Immune response to infection L15: Vaccines 2 Immune response to vaccination and infection L15: Vaccines 3 Types of Vaccines Live attenuated vaccines (e.g. measles, mumps, polio-Sabin, yellow fever) Viruses that have been weakened L15: Vaccines 4 Killed (inactivated) whole virus vaccines (e.g. Polio-salk; Vaccine to Japanese encephalitis virus, Rabies vaccine) Very safe, cannot replicate, completely inert Killed (inactivated) sub-unit virus vaccines (e.g. “split” vaccine to influenza) No longer infectious Recombinant vaccines Subunit - (e.g. hepatitis B vaccine) Simple protein vaccines Live, recombinant (chimeric) vaccines (e.g. DEN, JEV, and WNV) DNA vaccines (WNV) A lot of use in generating vaccines for cancer mRNA vaccines - COVID-19 Might not have had them without COVID Adjuvants (non-living viruses) Enhance the magnitude, breadth and durability of the immune response to vaccines Alum salts licensed in 1920 No new adjuvants for the next 70 years Hepatitis B, diptheria, tetanus and pertussis MF59 (oil-in-water emulsion) Licensed in Europe in 1990 for Fluad (older adults) Adjuvant system (AS) developed by GSK CpG 1018 used in Heplisav-B GSK - Adjuvant system L15: Vaccines 5 Adjuvants - not all about chemicals L15: Vaccines 6 Living vs. Non-living vaccines Live Attenuated Vaccines L15: Vaccines 7 L15: Vaccines 8 Vaccine safety L15: Vaccines 9 The cold chain transferring vaccines that must be kept at cold temperatures to remote locations Naked DNA Vaccines L15: Vaccines 10 Concerns with Naked DNA Vaccines Do DNA vaccines induce anti-DNA antibodies? Do DNA vaccines integrate into the host cell? L15: Vaccines 11 Use of Vaccines SARS-CoV-2: Vaccines 97 vaccines in clinical development 268 vaccines in pre-clinical development Incredibly fast moving Vaccines approved for use in people in less than 1 year Novel vaccination platforms have been approved No shortcuts to vaccine safety Close on going monitoring of approved vaccine Careful risk benefit analysis performed Approved Moderna - Spike mRNA Pfizer/BioNTech - Spike mRNA L15: Vaccines 12 AstraZeneca - Spike expressing Adenovirus Johnson and Johnson - Spike expressing Adenovirus mRNA vaccines Pfizer and Moderna COVID-19 vaccines granted emergency approval December 2020 95% effective at preventing severe disease Side effects Temperature limitations Advantage Disadvantage - Rapid R&D, simple production process - mRNA vaccines do not requrie nuclear - mRNA is unstable and easily degraded localisation signals and transcription - Strong immunogenecity, triggering in vivo unnecessary immune response - Safety is lower than inactivated - No risk of integration into DNA vaccines - Effectiveness is higher than inactivated vaccines mRNA vaccine design 5’ methyl cap Stabilises mRNA (RNase degradation, Alkaline phosphatase) 5’ Untranslated region Increases mRNA translation efficiency Coding sequence pseedouridine Reduces Toll-like receptor detection 3’ untranslated region Increases translation efficiency Poly(A) tail L15: Vaccines 13 Pfizer 120nt 3’ poly(A) tail mRNA vaccine delivery Carriers are required for mRNA entry into cells Packaged into Liposome Nanoparticles mRNA vaccine immune response L15: Vaccines 14 Needle free vaccine: Vaxxas High Density Microarray patch (HD-MAP) POlymer HD-MAP (patch) Cutaneous delivery, targets APCs to enhance immune responses Dried vaccine formulation to eliminate/reduce cold chain Short application time No needle/syringe Potential for self administration Virus neutralisation Viral Neutralisation: L15: Vaccines 15 Virus neutralisation of BAL fluid measured via PRNT against SARS-CoV- 2 virus isolates: 614D (wild-type, reference strain) 614G (contains G at residue 614 - now dominant variant) Higher neutralisation in HD-MAP groups Good neutralisation of 614D, 614G and B.1.1.7 isolates Protection from lethal SARS-CoV-2 challenge 1-dose groups 50% survival in unadjuvanted groups 100% survival and protection from weight loss and clinical signs of infection in adjuvanted groups 2-dose groups: L15: Vaccines 16 100% survival and protection from weight loss and clinical signs of infection in both unadjuvanted and adjuvanted groups No virus detected in lungs or brain Practice Questions 1. Define a vaccine and how it works? 2. What are the different types of viral vaccines – briefly describe each type, how they are produced and give examples? 3. Briefly describe the main properties of a naked DNA vaccine and how it works. L15: Vaccines 17 L16: Viral Diagnostics Microbiology & Immunology Courses (MICR2000) Table of contents Learning objectives - to understand Clinical examination Laboratory testing Virus isolation Viral neutralization assays Viral nucleic acid detection** Serology Immunofluorescence Antigen detection Lateral flow POC tests Point of care diagnostics Dengue infection Resurgence of Dengue and Dengue Hemorrhagic Fever Clinical significance of Viral Load in Dengue infection Kinetic biomarkers in dengue infection NS1 and antibody detection Practice questions Learning objectives - to understand Methods of diagnosis available Choice of test Types of lab tests available Why use lab diagnostic tests How tests are performed L16: Viral Diagnostics 1 Clinical examination Many viruses present with similar symptoms For a conclusive diagnosis, laboratory methods are required Laboratory testing Conclusive results obtained in hours-days Multiplex assays: testing for many different diseases at once Fast accurate diagnosis is critical for effective patient management E.g. Influenza antivirals are effective only if taken within 48 hours of symptom onset High specificity and sensitivity Low rates of false positive and false negative results Sample collection L16: Viral Diagnostics 2 Blood Saliva Nasopharyngeal aspirates Stool sample Swabs Spinal fluid Urine Choice of test? L16: Viral Diagnostics 3 Virus isolation Gold standard test for viruses Cells infected with sera or plasma collected early during the acute period of infection The presence of antibody can interfere with isolation Virus growth monitored by cellular morphology changes and cytopathic effect Isolate can be further characterised Plaque assay: take isolated virus, put it on the cells, do a 10-fold titration series, pick an individual plaque and sequencing to determine what it is Very accurate but takes a long time Viral neutralization assays L16: Viral Diagnostics 4 Commonly used to evaluate vaccine effectiveness Antibodies interfere with a virus’ ability to infect cells (neutralizes them) e.g. direct binding of an antibody to a viral particle L16: Viral Diagnostics 5 Viral nucleic acid detection** PCR based detection → amplifies signal Reverse transcription (RT) step for detection of RNA viruses Test for multiple agents in a signal assay (multiplex) Rapid High sensitivity and specificity Serology Detection of pathogen specific antibodies IgM: first response to pathogen appears 3-5 days post exposure. Marker of current or recent infection IgG: High affinity pathogen specific antibody, appears from ~7 days Marker of past infection or secondary infection Serology: ELISA detection L16: Viral Diagnostics 6 Immunofluorescence Antigen detection L16: Viral Diagnostics 7 Secreted viral proteins Surrogate marker of infection Fast, easy to perform, and reproducible Quantitative Lateral flow POC tests Point of care diagnostics L16: Viral Diagnostics 8 Performed at the point of care At the doctors surgery Assay time 15-20 minutes Require little or no sample processing. Can accept whole blood No specialised quipment required to read results Can be read by eye Qualitative: samples are positive or negative Dengue infection Disease resulting from dengue infection subclinical infection Self limiting febrile illness dengue fever dengue hemorrhagic fever Dengue shock syndrome Life long immunity to infecting serotype Infection with heterologous serotype can result in increased risk of DHF/DSS Half the worlds population are at risk of infection Tropical and subtropical regions of the world 390 million infections annually 500,000 cases DHF/DSS 22,000 deaths L16: Viral Diagnostics 9 Resurgence of Dengue and Dengue Hemorrhagic Fever L16: Viral Diagnostics 10 Clinical significance of Viral Load in Dengue infection Severity of dengue correlates with the viral load detected in patient serum early during the acute phase of secondary infection Both higher viral load an dincreased levels of the circulating viral protein, NS1 are found in patients who go on to suffer from more serious vascular leak that is characteristic of DHF/DSS Kinetic biomarkers in dengue infection L16: Viral Diagnostics 11 NS1 and antibody detection Combined NS1 and antibody detection increases the sensitivity of dengue diagnosis Confirm primary or secondary infection L16: Viral Diagnostics 12 Practice questions 1. Describe direct and indirect fluorescent assays 2. What dose the presence of a influenza specific IgM indicate in a patient sera? 3. What dose the presence of both IgM and IgG indicate in patient sera? L16: Viral Diagnostics 13

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