Unit 2 Flashcards

Question

Enveloped vs unenveloped viruses:

Answer 1

Enveloped viruses: - Acquire envelope as they bud through the host cell membrane - Viral envelope contains host cell lipid bilayer as well as viral proteins - Viral proteins contain receptors needed for virus entry Unenveloped: - Naked viruses are released by lysis of the infected cell - Viral receptors are present on the capsid surface

Answer 2

- More fragile than viruses with just a capsid - More easily destroyed by Disinfectants Detergents Outside environment - If the envelope is destroyed, then the virus is not infectious --> destroys the receptors needed for entry

Answer 3

UNENVELOPED VIRUSES Components: - Protein Properties: - Environmentally stable to: --> Temperature --> pH --> Proteases --> Detergents --> Drying Consequences: - Resistant to detergents - Can dry out and retain infectivity, are spread easily (aerosols) - Can survive adverse conditions in the gut - Lyse cell to release; therefore has to kill the cell. Usually cause acute infections ENVELOPED VIRUSES Components: - Lipids, proteins, glycoproteins Properties: - Environmentally liable to be destroyed by: --> Acid --> Detergents --> Drying --> Heat Consequences: - Easily destroyed by detergents - Must stay wet. Not easily spread (large droplets, secretions, transplants/transfusions) - Cannot survive in the gastrointestinal tract - Released by budding: Does not need to kill the cell to spread Can cause persistent infections

Answer 4

DNA viruses are more stable, show very little variation RNA viruses are more variable - RNA polymerase is error-prone and has no proof reading - Can adapt easily to new environments (jump species/zoonotic) Some RNA viruses are segmented so can reassort or swap genes

Answer 5

Nature of the viral genome - DNA or RNA; polarity of nucleic acid - Structure of nucleic acid (ss or ds); linear or circular; segmentation Structure and symmetry of the viral nucleoplasmid - Icosahedral; helical; complex Presence or absence of an envelope - Size and morphology - Genome organisation and different coding strategies - Tissue and cell tropism - Varying pathogenicity

Answer 6

STRUCTURAL PROTEINS: - Capsid proteins - Envelope proteins - Matrix protein (layer inside the envelope and outside capsid) - Virion associated enzymes NON-STRUCTURAL PROTEINS: - Proteins that are not structural components of the virus - Often enzymes (but some enzymes can be structural) - Also some viruses encode regulatory proteins, oncoproteins, etc.

Answer 7

The structural component of the virus capsid (icosahedral and helical and some complex viruses) Protect viral nucleic acid and deliver the viral nucleic acid to the cell Capsids of naked viruses contain receptors that attach to the host membrane to allow entry Contains sites that will induce an antibody response

Answer 8

Contain receptors that allow the virus to attach and then enter the host cell Are targets of the humoral and cellular immune response - Antibodies will recognise these surface exposed viral proteins Interact with the capsid during virus assembly

Answer 9

Proteins that are NOT structural components of the virus particle Made in the virus-infected cell following infection: - Often enzymes involved in viral replication ---> Proteases ---> Helicases ---> Polymerase (can be a structural protein) ---> Protein primers for nucleic acid replication - Can be proteins that help the virus avoid the host immune response - Targets of the host cellular response (T cell epitopes)

Answer 10

They can be transmitted inside vesicles (so sometimes have an envelope!!) Some RNA viruses can be transmitted as virus clusters inside vesicles Rotavirus and noroviruses are transmitted as clusters Vesicles remain intact (?) and they pass through the GI tract to the intestines

Answer 11

To enter a host, enter and grow in the right cells for them Produce more copies of itself Spread and infect more cells/another host

Answer 12

Virus enters host cell and grows, replicates and spreads May cause damage to cells and tissues --> Some viruses do not cause disease Pass onto new cells of the same or different type to produce the clinical effects we see

Answer 13

The ability of an organism to cause disease Composed of two factors: - Infectivity (ability to infect and colonise a host) ---> Measured as infectious dose (number of microbes necessary to initiate infection and cause disease) ---> E.g. Ebola virus can cause disease if only a few virus particles enter, while other viruses may require 10,000 particles to cause disease - Virulence (ability to cause host cell damage) ---> Occur along a spectrum ---> Pathogenic organisms always cause disease while less virulent forms may not cause disease at all

Answer 14

1. Attachment 2. Entry - Receptor-mediated endocytosis - Cell membrane fusion 3. Uncoating - At the plasma membrane - In endosome by changes in pH 4. Viral gene transcription 5. Genome replication 6. Translation 7. Assembly 8. Release - Non-enveloped viruses by cell lysis - Enveloped viruses by budding from the plasma membrane

Answer 15

Attachment to the host cell is a highly specific process - Involves complimentary receptors on the surface of susceptible host cells - highly specific - Receptor can be protein or carbohydrate - Initial binding is reversible - May cause a conformational change that then allows binding to a co-receptor

Answer 16

Viruses must cross the plasma membrane to enter the host cell Entry into the cell by either: - Cell membrane fusion (non-endocytotic pathways) - Receptor-mediated endocytosis (endocytotic pathways)

Answer 17

Virus membrane fuses with plasma membrane and nucleocapsid is released into cytoplasm Occurs at neutral pH (pH independent fusion) Examples: HIV, Herpesvirus

Answer 18

Virus particle binds to host cell receptors Enters cell in an endosome Virus membrane fuses with the membrane of the end-some and nucleocapsid is released into the cytoplasm

Answer 19

Cell membrane fusion 1. Initial binding gB or gC to heparin sulphate (a complex carbohydrate expressed on the surface of many cell types) 2. Attachment of gD to: - HveA (lymphocytes, epithelial cells, fibroblasts) - Nectin 1 and 2 (neutrons, epithelial cells, fibroblasts) 3. Fusion of the viral envelope with the cell membrane Uses multiple types of spike glycoproteins to bind and enter different types of cells

Answer 20

Cell membrane fusion 1. Binding of HIV gp120 to CD4+ T cells - Induces conformational change in gp120 2. Enables binding of gp120 to CCR5 or CXCR4 - Causes the gp120 trimer to break apart - Allows gp41 to be pulled towards the cell membrane 3. Fusion of gp41 with cell membrane - Releases nucleocapsid into the cytoplasm 4. Nucleoplasmids are targeted to the nucleus

Answer 21

Receptor-mediated endocytosis: 1. Binding of haemagglutinin (HA) to sialic acid receptor 2. Internalisation in clathrin coated pit 3. Movement into endocytotoic vacuole which fuse with lysosomes 4. Low pH triggers conformational change in HA trimer 5. Exposes fusion domain which allows the fusion of viral membrane and endosome membrane 6. Release of nucleoplasmids into cytoplasm

Answer 22

The release of viral nucleic acid from viral capsid Process is variable: For some viruses - Nucleic acids may still be in a nucleoprotein complex - The capsid is only partially disintegrated

Answer 23

DNA VIRUSES - Ds DNA viruses use host machinery in the nucleus (except poxviruses) to make more ds DNA - Ss DNA converted to ds DNA then replicates like ds DNA RNA VIRUSES - Replicate in the cytoplasm (except influenza and retroviruses) All make viral mRNA which then migrates into the cytoplasm to synthesise viral proteins using the host ribosomes

Answer 24

Translation of viral proteins in the cytoplasm Assembly of virus capsids from newly synthesised components (de novo assembly) Encapsidation of the viral nucleic acid

Answer 25

1. ENVELOPED VIRUSES Enveloped viruses are released by budding from the plasma membrane. Acquire the envelope on the way out from plasma membrane of internal membranes (nucleus, ER) 2. Non-enveloped viruses released by cell lysis

Answer 26

Viruses must overcome innate defences to enter the body - Both physical and immunological Many viruses enter via mucosal surfaces Different viruses adopt different strategies - Some viruses may use several entry routes ---> E.g. foot and mouth disease virus inhaled (most common) or ingested - Entry route may differ in different host species ---> E.g. influenza: Faecal-oral in wild birds and respiratory in humans

Answer 27

- Skin - Respiratory tract - Alimentary tract (GI tract) - Urogenital tract - Eye Viruses attach to cells at these locations by attaching to receptor molecules on certain cells

Answer 28

- The skin is an effective barrier (keratinised) - It must be breached by abrasions or bites - Macrophages, neutrophils, dendritic cells, natural killer cells

Answer 29

Some viruses remain localised at the site of infection - Influenza virus in the respiratory tract - Rotavirus in the alimentary tract Replication occurs in epithelium at initial infection site Cell-to-cell spread occurs, but virus does not disseminate to other tissues Usually acute (short incubation period, short duration) Site of shedding = site of entry

Answer 30

1. DEFENCES Specialised ciliated epithelium and mucus: mucociliary escalator (in upper respiratory tract (URT) and bronchi) - Filters out large particles (particles <5um can enter terminal airways and alveolar) Sneezing and coughing Innate immunological defences (e.g. alveolar macrophages, complement, cytokines, natural killer cells) 2. VIRUS ENTRY Via aerosolised droplets expelled by an infected individual - Spread by coughing or sneezing - Contact with saliva from an infected individual 3. EXAMPLES OF VIRUSES ENTERING VIA RESPIRATORY ROUTE: Influenza Foot and mouth disease virus Rhinovirus (common cold)

Answer 31

1. DEFENCES Low pH in stomach (denatures protein and kills most microorganisms) Bile and proteolytic enzymes in intestines - High pH in the duodenum (rapid change) Mucous 2. VIRUS ENTRY Oral route (ingestion) 3. EXAMPLES OF VIRUSES ENTERING VIA THE GI TRACT Rotavirus Norovirus

Answer 32

Some viruses can spread to distant sites Virus reaches blood via lymphatic system Primary viraemia (clinically silent - increases virus levels allowing infection of distant organs) Secondary viraemia (virus replication in other organs leads to high concentration of virus in circulation) Infection spreads to more sites Allows entry and exit routes from host to differ Usually have longer incubation period (more severe pathology) Greater involvement of adaptive immune responses and IgG

Answer 33

Entry: Bite of a rabid animal or contamination of scratch wounds by virus-infected saliva Rabies replicate in peripheral tissues (striated or connective tissue) at the site of infection Can remain at site of infection for days/weeks or longer Virus enters peripheral nerves (infects unmyelinated nerve endings in muscle) Spreads to CNS and enters the brain (causes behaviour changes) Migrates to salivary glands (replicates) and excreted in saliva No viraemia Also allows rabies to evade the immune system

Answer 34

Susceptible: - Appropriate cell surface receptors for entry (susceptibility) Permissive: - Able to support replication of the virus - May need particular cellular proteins to complete infection - May need to be in a particular cell type ---> Eg. canine parvovirus needs rapidly dividing cells - Specificity of a virus for a particular host, tissue or cell - Determines the host range of virus THIS IS VIRUS TROPISM

Answer 35

Not just receptors that determine tropism Cells need to be susceptible (able to support replication of the virus) There are other factors: - Cellular protease can activate fusion - Protease cleavage by digestive enzymes - Temperature of replication - pH lability of viruses - Anatomical barriers

Answer 36

Some enveloped virus require proteolytic cleavage of envelope glycoprotein for activation of fusion domain - Infectious virus is only found in cell types that contain proteases that cleave the glycoprotein - Eg. Influenza haemagglutinin (HA) spike protein ---> Must be cleaved into HA1 and HA2 by cellular proteases

Answer 37

Reoviruses are activated into infectious virions by cleavage with digestive enzymes Cleavage of VP4 spike to VP8 and VP5 Conformational change permitting virus to bind to M cells in the gut

Answer 38

Most human viruses replicate at 37 degrees Celsius Upper respiratory tract has a lower temperature - about 33 degrees Celsius Rhinoviruses replicate efficiently at 33 degrees Celsius but poorly at 37 degrees Celsius This limits their ability to spread beyond the upper respiratory tract

Answer 39

Gastrointestinal tract presents a harsh environment - Acid pH of stomach - Alkaline pH of intestine - Destructive effects of pancreatic enzymes Not many viruses can survive this - Most respiratory viruses are inactivated if swallowed (the exception is adenovirus) - Rotavirus/caliciviruses can survive (unenveloped viruses)

Answer 40

Ability of virus to breach barriers such as blood brain barrier will limit their distribution - Poliovirus/West Nile virus - Sometimes (but not always) spread to CNS

Answer 41

Fenner's pioneering experiments with mouse pox in the 1940s Mousepox can cause severe skin rash in mice Contact transmission Is this skin-to-skin transmission? How did Frank Fenner find this out? --> First study of serial daily titration of virus content of various organs and tissues to trace where the virus went in the body

Answer 42

Inoculated groups of mice in the food pad with mouse pox (caused lesions in the skin) At daily intervals looked for virus in: - Inoculated foot pad - lymph nodes - Spleen - Skin - Blood

Answer 43

Pathogenesis is how a disease develops in an organism, involving interactions between the pathogen and the host's immune system. It encompasses the steps from initial infection to symptom manifestation, influenced by factors like pathogen virulence and host immunity. Understanding pathogenesis is crucial for disease prevention and treatment strategies.

Answer 44

Scientific interest Control of virus diseases Diagnosis Sources of infection/ transmission routes: - Faecal-oral; respiratory - Aid disease control by reducing/preventing transmission Design effective vaccines: - Stop viraemia - No viraemia - many enteric viruses - vaccine design more difficult as injectable vaccines are not good at inducing IgA - Rabies - stop virus getting to CNS

Answer 45

DETECTION OF VIRUS OR VIRAL ANTIGEN: 1. In clinical samples: - Electron microscopy (virus particles) - ELISA to detect viral antigens/virus - Haemagglutination assay 2. In virus-infected tissues (by taking swabs/biopsies/ aspirates) - Immunoperoxidase assay - Immunofluorescence assay VIRAL GENOME DETECTION: - RT-PCR / PCR - Hybridisation

Answer 46

Shows the morphology of viruses --> can be used for characterisation and identification Can be performed on specimens directly or viruses concentrated from: - Faeces (rotaviruses, calicivirus) - Vesicle fluid (herpes simplex) - Skin scrapings (papillomavirus) Fast - can be done in a few minutes Excellent method for detecting rotaviruses, adenoviruses, astroviruses, caliciviruses

Answer 47

- Expensive - Need specialist equipment - Requires skilled personnel - Low sensitivity - Need concentrated virus samples (10^6 particles/ml)

Answer 48

Useful when numbers of virus particles is low Sensitivity and specificity can be improved by using virus specific antibodies Different viruses with similar morphologies can be identified

Answer 49

The sample is mixed with antibody Negative staining of the sample Loaded onto EM grid and visualised

Answer 50

Grid is coated with antibody and used to capture virus particles The virus sample is loaded onto antibody-coated grid Negative staining etc

Answer 51

1. Antibody coated well (capture Antibody). Well must be "blocked" with XS protein 2. Add sample containing Antigen 3. Wash well. Add antibody that recognises the Antigen (detecting antibody) 4. Wash well. Add conjugate labelled secondary antibody (and IgG peroxidase) 5. Wash well. Add substrate 6. IF POSITIVE: Colourless liquid --> coloured liquid

Answer 52

Adenoviridae Orthomyxoviridae Paramyxoviridae

Answer 53

Influenza virus contains an envelope glycoprotein called haemagglutinin which binds to red blood cells

Answer 54

PRINCIPLE: Red blood cells (RBC) --No virus--> RBC are not agglutinated and form a tight pellet at the bottom of the well Red blood cells (RBC) and virus ---> RBC bind to virus i.e. haemagglutination occurs and virus "cross-links" the RBC. Forms a shield PROTOCOL: 1. Virus sample diluted in 2 fold dilutions across plate starting with 1/10 dilution 2. Red blood cells (RBC) added to each well including control well (C = no virus) 3. Incubation at room temperature for 1 hour 4. RBC in control well should not agglutinate but will form a button at bottom of well. 5. In presence of virus, RBC will bind to each other i.e. agglutinate forming diffuse lattices that coats the well.

Answer 55

1. Cells from the clinical specimen are fixed onto glass slide 2. Add virus specific 3. Add a labelled antibody (fluorescent dye) that binds to the virus specific antibody 4. View with fluorescent microscope Used commonly for respiratory viruses in respiratory specimens

Answer 56

Similar to immunofluorescence except that a peroxidase label is used. 1. Cells from the clinical specimen are fixed onto glass slide 2. Add virus specific 3. Add a labelled antibody (peroxidase label) that binds to the virus specific antibody 4. View with microscope

Answer 57

Virus quantification Determine levels of virus (titre) in tissues by titration - Virus spreads to adjacent cells causing damage and death - Plaques are produced - regions with no cells, which can be seen with the naked eye after staining - Virologists equivalent of bacterial colonies - Used to quantify viruses ---> Virus titre = exact number of plaques ---> LD50 = dilution of virus that kills 50% of cells

Answer 58

Monolayers of cultured cells are incubated with serial dilution of virus to allow adsorption to cells After removal of the inoculum, the cells are overlaid with semi solid media (agar) which limits the spread of the virus to neighbouring cells Hence, each infectious particle produces a circular zone of infected cells which damage or kill the cells resulting in a plaque. Only viruses that cause visible damage can be assayed in this way Stain with crystal violet (stains living cells) to enable counting the visible plaques

Answer 59

1. Ten fold dilutions of the virus sample are made 2. One ml of each dilution is added to columns 1 to 5 (4 replicates) 3. The virus was grown for 4 days under medium containing agar 4. After 4 days, the cells were stained using crystal violet and plaque growth visualised 5. Virus titre in "plaque forming units" PFU per ml can be calculated from this: Virus titre = number of plaques (10) / number of replicates (4) x dilution factor = 2.5x10^7 PFU / ml

Answer 60

1. Nucleic acid purification (RNA or DNA) 2. Reverse transcription for RNA virus 3. Polymerase chain reaction (PCR): ---> Using virus-specific primers to amplify a specific target ---> Repeated cycles of denaturation, annealing and elongation 4. Agarose gel electrophoresis to visualise PCR products

Answer 61

- Looking for signs of damage - Detection of antibody - Viral cytopathic effect (CPE) observed in vitro - Cell culture

Answer 62

Look for signs of damage in: - Cells infected with virus in vitro ---> cytopathic effects observed in virus-infected cells ---> Type of damage caused by some viruses can be used by virologists for diagnosis - Experimental infection of animals

Answer 63

Antibody can be detected by: - ELISA - Immunofluorescence/ Immunoperoxidase - Haemagglutination inhibition assay - Plaque inhibition assay

Answer 64

Grow cells in vitro and infect with viruses taken from clinical samples Some viruses kill the cells they infect and as more cells are infected these changes can be visualised with a light microscope --> Easier to see this than the viruses themselves (too small) Range from massive damage to no visible damage (non-cytopathic) If CPE is not easily visible, can look for viral antigens in cells by immunofluorescence or immunoperoxidase

Answer 65

Sample from infected animal is used to infect cell culture in vitro: - Primary cell culture - Semi continuous - Continuous cell lines

Answer 66

1. Tissue fragments (cut with scissors/scalpel) 2. Treated with trypsin or collagenase 3. Cell suspension 4. Addition of liquid media (Eagle's and animal serum) Incubates in petri dish or tissue culture flask 5. Cells attach to solid surface and start dividing 6. Primary cell structure!

Answer 67

PRIMARY CELL LINES: - Prepped directly from animal tissue - Subcultured only 1-2 times - Technically more difficult - Best cell culture system as it supports wide range of viruses - Used when the state of cell differentiation is important - Difficult to obtain a reliable supply - Expensive EXAMPLES - Monkey kidney - Human embryonic amnion CONTINUOUS CELL LINES: - Derived from tumours or by treatment of primary cell culture with a tumour virus or mutagenic chemical - Can be propagated indefinitely - Most easy to handle - Range of viruses that can be grown is limited - Often do not resemble the original cell: ---> Less differentiated (lost morphology and biochemical features they possessed in the organ) ---> Often abnormal in chromosome morphology and number ---> Can be tumourgenic (cause tumours in mice when inoculated) EXAMPLES: - Hep-2; human epithelial - HeLa; Henrietta lacks, Human cervical cancer. Used to propagate polio - Vero; African Green Monkey Kidney cells

Answer 68

Confluent cell monolayer Epithelial and fibroblastic cells attach to plastic surfaces to form a monolayer Cells grown in a chemically defined medium Double in 24-48 hrs Retain viability after freezing at low temps (-70 to -176 degrees celsius) Cell cultures vary greatly in their susceptibility to different viruses

Answer 69

Fused cells containing many nuclei

Answer 70

Virus factories in nucleus or cytoplasm

Answer 71

Nuclear shrinking (pyknosis) in Picornaviruses Proliferation of nuclear membrane in Alphaviruses and Herpesviruses Vacuoles in cytoplasm in polyomaviruses Synctia (cell fusion) in Paramyxoviruses and Coronaviruses Margniation and breaking of chromosomes in Herpesviruses Rounding up and clustering of cells (no shrinking, little detachment) in Adenoviruses Rounding up and detachment of cultured cells in Herpesviruses, rhabdoviruses and picornaviruses

Answer 72

Virions in nucleus in Adenoviruses Virions in cytoplasm (Negri Bodies) in Rabies Virus "Factories" in the cytoplasm (Guarnieri bodies) in Pox viruses Aggregation of inclusion bodies around ribosomes in Arenaviruses Aggregation of inclusion bodies around chromatin in the nucleus in Herpesvirus

Answer 73

Inhibition of host cell biosynthetic machinery Toxic viral proteins Damage to cell membranes / cytoskeleton Lysis - Exit of non-enveloped viruses Apoptosis

Answer 74

What is the innate immune system and what is its function? What cells are involved in generating the innate immune responses? What viral products are recognised? What host receptors are involved? What is/are activated and what do they do (in general terms)?

Answer 75

The first line of defence against viral infection ---> Within hours of infection Provides rapid protection from viruses until the adaptive immune responses are induced - Broad specificity - No antigen processing required - No memory recall - Short duration Set the stage for virus-specific immune responses

Answer 76

Neutrophils (phagocytose bacteria and other pathogens) Antigen-presenting cells (APCs) (phagocytose the pathogen) - Macrophages + dendritic cells Natural killer cells (non-specifically kill virus-infected cells) Eosinophils (for parasites) Hold infection in check during the induction of specific acquired immunity

Answer 77

The first and predominant immune cell recruited in viral infection - Depletion of neutrophils during influenza virus infection leads to a more severe disease in mice. HSV infection results in neutrophil-depleted mice results in: - Increased viral load - Increased mortality (in mouse model)

Answer 78

Cells that rapidly seek out and destroy virus-infected cells (not specific) Identify markers of stress on the infected cells Has two types of receptor: - Activating receptor - Inhibiting receptor (prevents NK cells from killing healthy cells)

Answer 79

Capacity to kill virus-infected cells Bind to infected cells: - Release perforins - Induce apoptosis - Release interferon gamma Non-specific No memory No MHC restriction

Answer 80

Involved early in the host response Carry immunoglobulin Fc and C3b receptors (promote phagocytosis) Professional antigen presenting cells MHC restricted Initiate the adaptive immune response

Answer 81

Innate immune responses recognises pathogen-associated molecular patterns (PAMPs) Un-methylated C-G dinucleotides (CpG motifs) on DNA viruses --> High frequencies in viruses --> Low frequency in mammalian cells Double-stranded RNA (only RNA viruses produce this) Uracil-rich, single-stranded RNA

Answer 82

Pattern recognition receptors (PRRs) 1. Toll-like receptors (TLRs) - Found on the surface of cells and within endoscopes of phagocytic cells - Detect extracellular viruses 2. Cytoplasmic PRRs - Found in the cytoplasm - Rig-1-receptors (RIG-1 and MDA-5) - NOD-like receptors (NOD2) - Cytosolic DNA sensors Bind to viral products and activate a signalling cascade that results in the synthesis of cytokines that block virus replication in the infected host

Answer 83

Recognise extracellular viruses Can be either: - On the surface of cells (TLR-1,2,4,5 and 6) - In the endosome (TLR-3,7,8,9) TLR-3 binds ds RNA TLR-7 binds uracil-rich ss RNA (e.g. HIV) TLR-8 binds ss RNA TLR-9 binds CpG motifs within viral DNA Sense the presence of viral nucleic acid and other conserved molecular components of invading pathogens

Answer 84

Cytoplasmic pattern recognition receptors RIG-1 - Retinoic acid inducible gene-1 - Recognises ssRNA MDA-5 - Melanoma differentiation association gene 5 - Recognises ds RNA

Answer 85

The most important of the broad host defences against viral infection Protects adjacent cells from infection Inhibition of viral replication Helps activate T-cell mediated immunity: - Activation of macrophages - Up-regulated MHC receptors on virus-infected cells

Answer 86

Produced by most cell types early in infection (innate response) Activate genes that have antiviral activities - dsRNA dep protein kinase R - Rnase L Helps stimulate MHC class I (enhance presentation of viral peptides to T cells) Activates Natural Killer cells Induces apoptosis

Answer 87

Involved in the regulation of nearly all phases of immune and inflammatory responses (both innate and adaptive) Produced by natural killer cells and T lymphocytes Enhances MHC expression on Antigen-presenting cells More important as an immunoregulator than as an antiviral agent - Enhances the cytotoxic activity of T cells, macrophages and natural killer cells

Answer 88

Humoral immune response (B lymphocytes) - Days - Antibody production Cellular immune response (T lymphocytes) - Days - Cytotoxic T cells (CTLs) CD8+ ---> Kill virus-infected cells ---> Cytokines that eliminated viral RNA - T helper cells (Th) CD4+ ---> Activate macrophages ---> Cytokines Virus-specific immune response recognises viral proteins and carbohydrates.

Answer 89

Eliminate virus Destroy virus-infected cells Prevent re-infection

Answer 90

Cells that are resident in the lymphatic tissue Respond to antigenic stimulus by producing and secreting antibodies Carry highly specific receptors that recognise viral epitopes Recognise Antigens in their native form (no processing)

Answer 91

1. Activates on binding to virus/antigen 2. Requires a signal from T helper cells 3. Clonal expansion of B cells producing a single antibody 4. Initially IgM, class switches to IgG 5. Some will become long-lived memory B cells Antibodies early on are low affinity (poor binding). B cells evolve by hyper-mutation in the V regions to produce high-affinity binding antibodies later on

Answer 92

IgA and IgM are detectable within 10-14 days then they decline IgG and IgA predominate in the secondary response

Answer 93

The main circulating antibody Important for long term immunity

Answer 94

Circulating antibodies produced early in infection Diagnostic marker?

Answer 95

Secretory antibody Primary defence at mucosal surface

Answer 96

Antibody binds to circulating virus (outside of the cell) - May prevent attachment to susceptible cells (neutralises virus) Opsonisation - Virus-coated with antibody activates complement - Induces inflammatory response (attracts phagocytes) Neutrophils and macrophages - Phagocytose virus coated with antibody Plays a major role in recovery following infection with viruses that cause viraemia

Answer 97

T-lymphocytes co-ordinate the activities of the cells involved in generating the immune response - T helper cells (CD4+ T cells) - Cytotoxic T cells (CD8+ T cells) Carry highly specific receptors that recognise specific regions on a pathogen (single T cell recognises a single T cell epitope) in association with MHC proteins On activation, T cells will divide and proliferate and secrete cytokines that regulate the immune response Some T cells become long-lived memory T cells

Answer 98

Recognise viral peptides on the surface of infected cells associated with MHC class 1 Kill virus-infected cells: - Granules within the CTL polarise towards the target cell - Perforin is released and creates pores in the cell membrane causing lysis

Answer 99

Recognise viral peptides on the surface of APCs associated with MHC class II Plays essential role in initiation of B cell responses Secrete cytokines that regulate the immune response Main types are Th1 and Th2

Answer 100

Th1: - Secretes IL-2 - Inflammatory response. Augments immune response by attracting macrophages to site of infection - Activates IFN y and TNF ß - Promotes IgG2a production Th2: - Secrete IL-4, IL-5 and IL-6 - Provide help for antibody production - Promotes switching of B cells (IgG2a to IgG1)

Answer 101

Surface exposed protein on virus particle: - Envelope proteins - Capsid proteins (of naked viruses) Often spikes or loops that protrude from viral surface Epitopes can be linear or 'conformational' (critical residues brought together by folding of the polypeptide chain)

Answer 102

Surface proteins are recognised by antibodies B cell epitopes are on surface exposed proteins and more likely to be "conformational" Internal (non-structural) and surface proteins are recognised by T cells T cell epitopes can be internal or external proteins Epitopes are linear (continuous)

Answer 103

VIRUS: Infects cell: Stimulates innate response Viral peptides are expressed on cell surface with MHC I ---> Activate CD8+ cytotoxic T cells --> Memory T cells Taken up by APCs: Macrophages and dendritic cells. Viral peptides expressed in association with MHC class II. - Activates CD4+ T cells ---> Th1 cells activate macrophages (back to taken up by APCs) ---> Th2 cells. Helps B cells (goes to next part) Drains to lymphatic tissue: Lymph tissue in gut (GALT) and lungs (BALT) tonsils/Peyers patches - Produce IgA Lymph nodes/Spleen - Receive virus from the blood circulation and lymph - Produce IgM initially and then switches to IgG ---> Memory B cells

Answer 104

Immunofluorescence (IF) ELISA Virus neutralisation assay Western Blotting (only rarely used) Measures only the humoral immune responses (antibody) Techniques for cellular immune responses are available for research, but are NOT routinely available or cost-effective for diagnostic testing.

Answer 105

Can be either: - Qualitative (positive or negative) - Quantitative (how much antibody) PROS: Rapid, cost-effective, easily scaled up Often doesn't require infectious virus (as recombinant proteins can be used) CONS: Species-specific (but can do competitive ELISA)

Answer 106

Need to determine the cut off for the ELISA - OD reading above which the sample is positive - Need to run several negative samples Cut off Mean of the known negatives (non-infected) plus 2 (or 3) standard deviations - Assumes distribution is normal Twice the mean of the negatives (non-infected) samples No consistent methods used Method is decided for each ELISA (empirically) Method that gives the best results with known positives and negatives is used

Answer 107

Perform the ELISA with: - Test samples - Positive and negative controls - Serial dilution of antibody standards (contain known concentration of antibody) - Draw a standard curve (antibody concentration vs OD reading)

Answer 108

Draw the standard curve: - Plot Log10 Antibody concentration on the X axis - Plot OD on the y axis Determine the concentration of antibody in the test samples by reading from the graph Above a threshold, the same will be considered positive - Determined empirically for each disease

Answer 109

1. Run proteins on a SDA polyacrylamide gel 2. Transfer separated proteins to a nitrocellulose/ nylon membrane 3. Incubate membrane with antibody (from sample i.e. diluted serum sample). Wash, incubate with secondary antibody labelled with enzyme/tag that can be visually detected 4. Detect/visualise proteins on addition of substrate

Answer 110

Allows detection of antibodies to some or all of the viral proteins of a particular virus Can be used to monitor the presence of antibodies to different antigens at different stages of infection Not used routinely in diagnostic setting (not easily standardised or scaled up for automation) Generally used as a confirmatory test

Answer 111

Loss of infectivity through reaction of the virus with specific antibody Virus is incubated with serum sample (serial dilutions) - Using the constant concentration of virus --> Concentration where plaques are visible and beginning to decrease in number - Virus and antisera are then used to infect cell monolayers in a microtitre plate - Determine the highest dilution of antibody which when mixed with a standard virus concentration will prevent the formation of plaques

Answer 112

Fixed concentration of virus in all wells Antibody is serially diluted across the plate Neutralisation titre can be determined

Answer 113

Sometimes titres represent the serum dilution (or the reciprocal of the serum dilution) giving 100% neutralisation against CPE compared with uninfected cells But different scientists can use different cut offs. Essentially the percentage plaque formation is calculated and plotted vs antibody dilution and then the titre is read off the graph

Answer 114

PROS: Used for detection and quantification of virus specific antibodies Gold standard in virology Neutralising antibody correlates with protective antibody in vivo When new virus emerges - such tests can be operational within weeks (take up to a year for an ELISA to be fully validated) CONS: Slow. Can take several days to get a result Require infectious virus Cost to maintain cell culture facilities

Answer 115

Used when it is difficult to detect virus directly Widely used when animals are imported and exported from countries - Governments want certification to ensure animals are not bringing pathogens with them Used to assess vaccine efficacy: - Is vaccination being implemented correctly - Eradication programmes

Answer 116

DISEASE: Respiratory disease in humans, horses and swine (and other mammals) Enteric disease in birds LOCATION: Replicates in upper respiratory tract in humans Replicates in the intestines and respiratory tract in birds HOST: Reservoir host is in birds Human, horses, dogs, and swine (and bats) Newly pathogenic strains emerge (increased tropism and jump species)

Answer 117

Member of the orthomyxoviridae family Enveloped virus (80-120nm) Negative sense, ss, RNA genome Segmented RNA genome (8) Helical nucleocapsid Most segments codes for a single protein (mostly!) - Haemagglutinin (HA) spike - Neuraminidase (NA) spike

Answer 118

Inside the virus: - NP (Nucleocapsid) - PB1, PB2, PA (transcriptase complex) - NS2 On the outer layer of the virus: - M1 (Matrix protein) - M2 (ion channel) - Lipid bilayer Protruding from the virus: - NA (Neuraminidase) - HA (Haemagglutinin) Carry important type-specific antigens: 18 H types 11 N types

Answer 119

Haemagglutinin (H): Human: 1,2,3 Equine: 3,7 Swine: 1,3 Avian: 1,2,3,4,5,6,7,8,9,10,11, 12,13,14,15,16 Neuraminidase (N): Human: 1,2 Equine: 7,8 Swine: 1,2 Avian: 1,2,3,4,5,6,7,8,9 New H17 N10 and H18N11 subtype found in bats

Answer 120

1. Virus enters the host cell 2. Virus replication occurs in the nucleus - Viral mRNA need to be capped at their 5' end (5'C) - Some viral mRNAs are spliced Capping and splicing enzymes are in the nucleus!! 3. Neuraminidase aids release of the virus from the cell 4. Spreads to other cells in the upper respiratory tract. Virus normally remains localised in the respiratory tract

Answer 121

Influenza viruses have the ability to evolve rapidly. Antigenic shift: - Complete change in subtype of HA or NA - Change in HA can result in pandemics Antigenic drift: - Variation within the HA or NA subtype - Drift in HA or NA results in epidemics and more rarely pandemics - Can also allow influenza to "jump" species - Can cause changes in virulence/tissue tropism (as seen with Avian influenza H5 and H7 subtypes) Allow virus to change species specifically and to change virulence.

Answer 122

Complete change in HA type Virus acquires a different HA type from a different species as a result of re-assortment of the viral segments Pigs generally act as mixing vessels as they can be infected with both human and avian viruses Causes pandemics Population are immunologically "naïve" Host immune system has not seen this gene before No protection

Answer 123

1918: SPANISH FLU - 20-40 million deaths H1N1 - Avian virus? 1957: ASIAN FLU - 1 million deaths H2N2 reassortment of human and avian viruses Avian HA (H2) Avian NA (N2) Avian PB1 1968: HONG KONG FLU - 1 million deaths H3N2 - Reassortment of human and avian viruses Avian HA (H3) and Avian PB1 1977: RUSSIAN FLU H1N1 Re-emerging virus 2009: SWINE FLU vH1N1

Answer 124

Classical H1N1 swine flu: - Emerged in swine in 1918 - Circulating for decades in US - Mutating slowly Human H3N2: - Emerged in humans 1968 Swine H3N2: - Emerged in 1997 Swine H3N2 + Avian H1N1 = Swine H3N2 Emerged in 1999 spread rapidly in swine + Classical H1N1 Swine Flu = Swine H1N2: - Occasional transmission (11 cases) to humans. - Usually severe Swine H1N2 + Eurasian swine H1N1 (emerged in swine in 1979) = New pandemic vH1N1

Answer 125

New swine influenza detected in a human patient First human case of this kind in the UK Identified as part of routine surveillance Patient experienced mild illness and recovered Source has not been identified: - Patient had no contact with pigs - Contact testing

Answer 126

Minor variations in the sequence of HA and NA genes Caused by mutation Causes epidemics

Answer 127

Variation in spike proteins present on the viral surface. Produce viruses with slightly different amino acid compositions Some may grow better in a particular host than others Gradual accumulation of antigenic mutations

Answer 128

Change in host (species specificity) Change in tropism

Answer 129

The most abundant protein on the viral surface and is the main target of immune response Receptor binding site: - Allows virus to bind to sialic acid on host cells - Enables virus internalisation (within endosome) Cleavage site: - Cellular proteases cleave HA into two subunits (HA1 and HA2) to allow virus release from the endosome - Cleavage aids fusion of the virus membrane with the membrane of the vesicle - How well HA is cleaved by cellular proteases facilitates virus entry

Answer 130

Attachment: - HA binds to sialic acid groups on membrane-bound proteins on the cell surface Entry: - Virus enters by receptor-mediated endocytosis Release from endosome: a) Acidic pH inside the endosome, causes a conformational change in HA, which exposes a fusion domain which promotes fusion of viral and endosomal membranes b) Viral nucleocapsids to be released into the cytoplasm

Answer 131

LOW PATHOGENIC AVIAN INFLUENZA: - Causes a wide range of symptoms in wild birds - Generally a mild illness - If the virus infects domestic birds, it can occasionally develop into a highly rapidly fatal disease or high pathogenic avian influenza HIGHLY PATHOGENIC AVIAN INFLUENZA: - Results in severe epidemics - Sudden onset - Severe illness - Rapid death (within a few hours) - Mortality can be up to 100% - All pathogenic forms are caused by H5 and H7 - Virus changes from a virus that is enteric to a virus that infects every cell in the body.

Answer 132

LOW PATHOGENIC AVIAN INFLUENZA: - HA1 and HA2 polypeptide chains linked by a single arginine residue - Cleaved by a few cellular proteases in a limited subset of cells - Therefore influenza normally can only replicate in a few cell types. HIGHLY PATHOGENIC AVIAN INFLUENZA: - Insertional mutations add basic residues at HA cleavage site - HA1 and HA2 polypeptide chains linked by several basic amino acid residues - Cleaved by ubiquitously expressed cellular proteases (Turin and other subtilisin family proteases) - Virus can replicate throughout bird's body producing necrotic foci in many places

Answer 133

Influenza binds to sialic acid on the surface of host cells Sialic acid is found on the end of polysaccharide chains How sialic acid is bound to galactose determines its shape

Answer 134

Sia(alpha)2, 3Gal - Recognised by HA of avian influenza e.g. H5N1 - Long thin structure Sia(alpha)2, 6Gal - Kinked shape - slightly wider - Recognised by HA of human influenza - E.g. H1 and H3

Answer 135

Sia(alpha)2, 6Gal - Present in the human upper respiratory tract - Therefore human viruses can enter and cause infection Sia(alpha)2, 3Gal - Present in the human lower respiratory tract - Therefore avian virus can enter and cause infection (but only when in close contact with avian viruses) Therefore, under "normal" circumstances the avian virus is restricted to birds

Answer 136

H1N1: Easily spread, rarely fatal H5N1: Spreads slowly, often fatal

Answer 137

Several mutations have been implicated: - Reduce binding to Sia(alpha)2-3Gal - Increase binding to Sia(alpha)2-6Gal - For H1/H3 - as few as 2 amino acids can switch human and avian receptor specificity

Answer 138

Required just 4 mutations in HA - three fro binding to Sia(alpha)2-6Gal (human HA receptor) - One for stabilising HA

Answer 139

Viral targets of humoral immunity - HA and NA - Antibodies to these proteins correlate with protection Antibodies to one subtype are NOT cross-protective Strain-specific immunity can be long-lasting Infection does NOT induce life-long protection (WHY?)

Answer 140

Neutralising antibodies target the globular head of HA - Neutralise virus by binding to or near the receptor for host cells - Therefore blocks binding and entry of virus to cell Are strain specific and lack cross neutralising antibody to different HA subtypes Are epitopes on the stalk/stem region (these are hidden) - Conserved amongst the different HA subtypes - Can be broadly neutralising against many HA subtypes but titres are low during infection

Answer 141

Neuraminidase (NA) plays an important role in allowing release of influenza from the cell After budding, NA cleaves sialic acid from the cellular receptors - Destroys the receptor and prevents reinfection of the same cell Antibodies to NA can block this enzymatic activity - Therefore do not prevent infection but limit virus spread - But do reduce replication by inhibiting the release of newly produced viral particles (reduce severity and length of infection)

Answer 142

CD4+ and CD8+ T cells are induced by: - Envelope proteins (HA and NA) - Internal proteins (NP, M1, PB1, PB2, PA and NS1) CD8+ T cells: - Eliminate virus-infected cells ---> Release perforin and granzyme ---> Release IFN gamma and TNF alpha - Epitopes are conserved within subtypes so may be cross-protective

Answer 143

One health is a comprehensive approach to health that focusses on: 1. Improving health and wellbeing through the prevention of risks and the mitigation of effects of crises (e.g. emerging diseases) that originate at the interface between humans, animals and their environments 2. Promoting multi- (cross-) sectoral collaborations and a "whole of society" treatment of health hazards, as a systemic change of perspective in the management of risk

Answer 144

Conference of Wildlife conservation society in New York introduced: - The term "One World One Heath" - 12 recommendations (Manhattan Principles) for preventing epidemic disease and maintaining ecosystem integrity for the benefit of people, domesticated animals and the foundation of biodiversity that supports us all.

Answer 145

Zoonotic disease, emerging disease, multi-host pathogens, food safety, food security (farming practices), antimicrobial resistance, mental health, obesity, climate change, etc. Applies across research, education, policy and practice

Answer 146

Growing recognition of need for interdisciplinary collaboration to prevent and control emerging zoonoses. E.g. among doctors, veterinarians, wildlife specialists, anthropologists, economists Berlin Principles (2020) update on Manhattan principles emphasising the need for action.

Answer 147

- COVID 19 - Highly pathogenic avian influenza (H5N1); the 2009 swine-origin influenza pandemic - Emerging zoonoses/infections (e.g. 2014 Ebola epidemic) - Climate change - Population growth and urbanisation - Challenge of providing food - Encroachment and contact with wildlife - Antimicrobial resistance (human and animal overuse) - Globalisation of trade and animal movement. It is estimated that 75% of recently emerging infectious diseases are of animal origin and 60% of all human pathogens are zoonotic. The class of pathogen most responsible for new human infections is viral because of their high replication and mutation rate.

Answer 148

An infectious disease that transmits from a non-human animal to humans

Answer 149

An infectious disease that transmits from humans to non-human animals

Answer 150

One that has either newly appeared in a population or that has a rapidly increasing incidence or expanding geographic range. Emergence is associated with (previously unrecognised) cross-species transmission (host jumping)

Answer 151

Biodiversity surveys reveal a myriad of viruses at the interaction zones between humans and animals.

Answer 152

Kinshasa's railway helped make it one of Africa's best connected cities A perfect storm of population growth, sex trade and connectedness allowed HIV to spread during the 1920s

Answer 153

- Human behaviour - Antimicrobial misuse - Globalisation, trade and travel - Conflict and political upheaval - Weak surveillance, control, health systems - Agricultural intensification - Water management - Climate change - Land use change - Population growth and urbanisation

Answer 154

One Health has a broad scope: Zoonotic and emerging pathogens are one element We live in a complex, inter-connected and altered world A key feature of One Health is that it integrates multiple disciplines to solve complex challenges Your biological and bioveterinary training is an excellent starting point for contributing to One Health

Unit 2 Flashcards

(178 cards)