Virus

Virus

1. Immuno...diagnostics

   • Reverse transcripitase PCR
     • Same principle as normal PCR. Useful for detecting mRNA quantities.
     • Can use reverse transcriptase and primers. Forms cDNA with primer and RNA. Only amplifies RNA - only till it is visible.
       • Electrophoresis occurs and we can see the DNA fragments. Helps locate where virus is. Intensities can allow the measurement of [RNA] and viral load. High = immediant antiviral
   • Only a few viruses are able to coagulate blood (viral haemagglutination). Some viruses have the property and this can be used to see how affected they are.
     • HI test - haemagglutinin Ihnibition Assay: Used to measure how well ab bind to influenza virus. RBC in a solution will sink to the bottom of the assay well and form red dot at the bottom
     • If virus is present, viruses haemagglutinin surface proteins bing to RBC and keep RBC suspended
   • Detection of host ab against virus: Tract the ab and see if they work against the pathogens
   • Molecular biology & diagnostic virology: genome.
     • Methods include: Viral DNA hybridiation with labelled probes. Sequencing or portions of the viral genome. Polymerase chain reactions
     • Modern techniques - protein chemistry and DNA/RNA biochemistry. Used for any infection. Efficient/Fast. Improve specificities of virus diagnostic
   • Presence of anti-virus ab in patients will inhibit the coagulation so RBC don't interact
   • Ab used to inhibit, detect and qualify virus. We combine ab with biomarker e.g. radiolabel or fluroscence
   • ELISA

2. Influenza

   • Influenza, there is lots of antigen-antibody reactions. Can see virus and severity.
   • Spanish influenza - severe strain of influenza killing more people than WWI
   • Reassortment - results in antigenic shift which can make hybrid strains and cause pandemics e.g. Corona or Spanish flu
   • Antigenic shift: Process of which 2 + different strains combine to form subtype with mixture of antigens. No longer recognized by the immune system
   • Transmission between species. Airborne disease which affects respiratory mainly. Difficult to diagnose due to symptoms ranging from mild to severe
   • Zoonotic disease - pathogen jumping from animal to human
   • Antivirals - Treatment in some case but more side effects. M2 ion channels are target for antivirals e,g Rimanatadine or Amantadine
     • Neuraminidase Inhibitors
       • Prevents the release of the virion. Oseltamivir is a competitive inhibitor of the activity of the viral neuraminidase. Prodrug is hydrolysed in liver.

3. Genome

   • RNA
     • ssRNA: tSense RNA used directly as RNA. Very fast replication
       • Orthomyxovirus: Inflenza A, B and C Rhinovirus - common cold
       • Togaviruses: Rubella - congenital rubella infection, intrauterine rubella infection, miscarriage, stillbirth, abortion
       • Paramyxovirus- Mumps, Picornaviruses - Hep A
     • dsRNA
       • Rotaviruses: group A - severe gastroenteritis in infant and young children
       • One strand of RNA used directly as mRNA
   • Retroviruses: Any group of viruses that belong to the family of Retroviridae and carry blueprint for RNA. Contsins unique enzymes to make DNA from RNA. Integrate gemone into host cells genome which means it is uncurable,
     • Single stranded RNA e.g. HIV
     • Double-stranded DNA e.g. Hep B
       • These are called reverse transcriptase viruses - RNA intermediate before viral proteins can be manufactured
       • First transcribe DNA into RNA bedore it is inserted into the host DNA.
       • Reverse transcripitase are enzymes used to encode retroviruses viral genome. Enzyme is responsible for transcriptase of the viral RNA to produce dsDNA that can be inserted into host genome.
   • DNA
     • dsDNA: makes mRNA ro make proteins to invade host cells
       • Adenoviruses: URTI. Has a dsDNA genome enclosed in an icosahedral capsid. Virus transmitted orally
       • Poxvirus: Variola - small pox virus, vaccinia - derived from cowpox virus, immunize against smallpox
       • Papillomavirus: Cervical cancer
       • Herpes viruses: HS1&2..
     • ssDNA: Makes a copy of the strand to make direct mRNA and the host cell makes ss or ds proteins
       • Parvovirus: B-19- fitfth disease slapped child disease. Symptoms include low grade fever, headache, cold light symptoms, rash commonly on face.
         • Incubation 4-21 days. Transmission via respiratory secretions - issues with pregnancy and immunocompromised patients

4. Viral classification

   • 1) Morpholgy: Size, shape, enevelope or not
   • Most commonly used classification is Baltimore classification scheme - based on how mRNA is generated in each type of virion. Obsereved by SEM and TEM
     • Baltimore classification: a classification scheme that groups viruses into 7 classes according to how the mRNA is produced during the replicative cycle of virus
   • 2) Physiochemical properties: Mr, thermal and ionic stability
   • 3) Genome: RNA/DNA
   • 4) Macromolecules: Protein composition and function
   • 5) Antigenic properties
   • 6) Biological properties: Host Range
   • Viruses can be double stranded DNA (dsDNA), single stranded DNA (ssDNA), doubled stranded RNA (dsRNA) and single stranded RNA with positive polarity or negative polarity (ssRNA)

5. Viral Capsid/ Nucleocapsid

   • Capsid denotes the protein shell that ebcloses the nucleic acid. It is built of structure units - formation doesn't require energy. It is very efficient and build quickly
     • Virus have all : definite shape, genes, ability to infect host cells and ultramicroscopic size
   • Individual proteins fit together like puzzle pieces to form a building block called a capsomere.
   • Virus building blocks proteins are assembled to form a tight shell called a capsid. Inside capsid nucleic acid genome lodges for protection
   • Caspid may take the form of a polyhedron (icosahedral) or it may be spiral (helical symmetry) or it may be more complex

6. Virus envelop

   • Viruses survive longer when mixed with fmutes: blood, musuc
   • Contains host proteins but also virus-encoded proteins. Allows immune system to recognize the antigens as own celsl
   • Surrounds the viral capsid in some but not all viruses e/.g HIV. Enveloped are susceptible to low desiccation as the envelope is very weak from physical and chemical challenges
   • Responsible for viral entry into host cells: Recognition of host cell receptors. Initiation of entry by fusion or endocytosis
   • Derived from portions of the host cell membrane (phospholipids and proteins) during viral replication. Cytplasmic membrane, cell nuclear envelope and organelle e.g ER
   • Budding:: Newly formed viral particle pushes against host cell membrane which adheres to it. Membrane envelops the virus to become a viral envelope. Virus is released from cells. Proces uses hiost's cell membrane and leads to cell death
   • Capsid is made up of proteins which surrounds the DNA /RNA and is surrounded by the envelope

7. What are virus

   • Acellular, carriers of DNA or RNA. Requires a host for replication. Obligatory intraceullar parasites. Known as pathogenic and difficult to treat without harming the host
   • Virus cannot multiply themselves so require cellular organisms to replcate their DNA = make new virions. Main purpose is to replicate parts as often as u can - exhausts cell resources while the burst of virions destroy cells
   • Virus differ greatly in size and morphology. 20nm to 240/400nm in diameter, Two types of virus: rods or filaments (linear arrays of nucleic acid and proteins) and spheres (20 sided icosahedral polygon)
   • Airborne: Chickenpox, Influenza, MMR, Smallpox
   • Direct contact: AIDS, Cold sores, HPV, Genitial herpes, Leukamia, Hep B and C
   • Food/Water Gastroenteritis. Norovirus, Hep A, Poliomyelitis
   • Arthropod: Yellow fever (indirect transmission from insect)
   • Cannot be seen under light microscope

8. HIV

   • Cell cycle
     • 1) Viral attachment
     • 2) Binding and fusing (fusion of HIV to CD4)
     • 3) Viral uncoating (capsid enters cells)
     • 4) Transcription and translation (Viral DNA formed)
       • 5) Integration (Viral DNA is used to make genomic RNA)
         • 6) Assembly (New viral RNA is used to make genomic RNA and proteins)
           • 7) Maturation and budding (Virion moves to the cell surface to form immature HIV form. Virus is released
   • Glycoprotein receptors which form the external spikes. Contains reverse transcriptase that are used by retroviruses
   • In HIV: there are four targets for antiviral
     • Entry: Fusion inhibitor receptor agonist
     • Reverse transcriptase: Noe 1 target
     • Viral Integrase: Stops integration of RNA into cell DNA
     • Viral protease: Ihibitis protease
   • Integrase enzymes integrate viral DNA into cellular DNA which is essential step. Allows it to integrate its genome into host without destroying the cell. Proteases therefore are important antivirals targets

9. HSV

   • Reactivation in neuronal cell body: 1) Initiation of viral lytic gene expression ................................................................. 2)newly formed capsids are transported to axonal termini .......... 3) Infections virus is released from axon to infect epithelial cells resulting in reccurent infection
   • Infection of epithelial cells in mucosal surface
   • HSV may persist in quiescent but persistent form = latent infection in neuronal ganglia
   • Productive, replication: production of progeny virions and virions spread to infect additional epithelial cells
     • Virus enters innervating sensory neurones and nucleocapsids are transported to neuronal cell body
       • Viral DNA is released into the neuronal nucleus and circularize
         • Circular Viral DNA persists in the neuronal cell nucleus
           • Viral genome penetrates neuronal cells which results in virus remaining even after symptoms are gone. Virus can reactivate and leave neuronal cells to form nw coldsores etc. Only a huge problem in immunocompromised patiwnts

10. Antiviral Chemotherapy

    • Integrase Inhibitors
      • Viral integrase: An enzyme that allows transfer of HIV cDNA to cellular DNA. Inhibiting this prevents this E.g Dolutegravir
    • Entry Inhibitors: Fusion Inhibitors
      • Fusion inhibitors: An enzyme that prevent HIV envelope fusion. Nornamlly it recognises each other and results in HIV envelop fusing around membrane of cell e.g Enfuviritide
        • Oseltamivir (Tamiflu) and Zanamivir (Relenza) are two types of antiviral medications used against the flu virus.
        • Anti-virals do not take the place of vaccination. Vaccination is the most effective way of protecting against flu.
    • NRTIs (Nucleoside reverse transcriptase inhibitors)
      • Reverse transcripitase - No 1. Most highly active anti-retrovirus therapy (HAART). Therapies contain RT-inhibitor . Used to treat HIV e.g Zidovudine or tenofovir
        • DNA is synthezised from 4 deoxynucleotide triphoshphate. AZT is a nucleoside analogue so it resembles the nucleotides.. So when DNA replication occurs AZT is added instead of normal base which haults the proces. It prevents further extensions of growing chain
    • Protease Ihnibitors
      • Viral Proteinase: An enzyme that cleaves peptides/polypeptides/protein chains. Polypeptides produced during viral replication is known as immature virus and then protease form a mature virus
      • HIV protease inhibitor bind to enzyme active site. HIV virus is unstable so process the proteins. E.g. Atazanavir
    • HSV: Antiviral Inhibitors of viral DNA polymerase
      • Viral DNA polymerase: enzyme that creates DNA molecules by assembling nucleotides - essential for DNA synthesis. Aciclovir is a guanosine analogue converted by viral thymidine kinase and again till ACV-TP. ACP-TP competitively inhibits and inactivates HSP=specific DNA polymerase
    • NNRTIs ( Non-nucleotide transcripitase inhibitors
      • Targets HIV reverse transcripitase too by attaching to the enzymes and affecting the activity - restriction mobility = unable to function. Causes congifuration change of active site so RNA cannot bind to enzyme. E.g. efavirienz/etravirine
    • Receptor antagonist
      • Entry inhibitor: A negative allosteric modulator of the CCR5 receptors. Which is a protein on the surface of WBC that is involved in IS as it acts as a receptor for chemokines

11. Viral Receptors

    • Glycoproteins (virus encoded) on the surface if envelope from capsid to identify and bond to receptors sites on the host membrane e.g haemgluttinin, Gp120

12. Diagnosis

    • Symtoms: Most common - fever
    • Viral isolation (direct): Immunofluorescence - probes detect and emit
    • Viral Antigen/virus: Haemagglutination Ihibition test, neutralisation, complement fixitation test
    • Loads of viruses are hard to diagnose so must be cultured. Monkey derived cells, 24 hr and viruse is grown
      • Plaque assay: 1) Grow a lawn of host cells 2) Add virus 3) Virus replicates and lyse host cells 4) Viral plaques: cytopathic effect
    • ELISA: Enzyme-linked immunosorbent assay - commonly used to analysed/detect the presence of a ligand in a sample using ab directed against proteins
      • Detects antiviral antibodies and use iab to identify a substance. Solid-phase of ELISA will recognize the ab and bind to it to form anti-human ab with enzyme conjugate. Detection system identify different colours produced. Sensitivies to concentration in patient serum can cause different intensities of ocloirs.
    • Antivirus antibodies and immunoflurescence
    • Produce a circular zone of infected cells known as plaque - dues can be used
      • One plaque = One virus. Units = PFU (plauq forming units
    • Biological specimen for viral : Nasopharynx swab/ aspirate, throat/lesion/eye swab, blood, slavia, tissue biopsy, urine, stool

13. Contact Inhibition: Virus stops dividing when in contract with other cells

14. Cytopathic effect: Ocurrs when infected cells causes lysis of cells or when cell dises without lyses dye to inability to reproduce

15. Bacteriophages

    • Bacteriophages are viruses that infect bacterias/archae bacteria. Contain tail fibres which help attach the virus to host cell receptors.
      • After that a phage usually follows one of two life cycles, lytic (virulent) or lysogenic (temperate). Lytic phages take over the machinery of the cell to make phage components. They then destroy, or lyse, the cell, releasing new phage particles. Lysogenic phages incorporate their nucleic acid into the chromosome of the host cell and replicate with it as a unit without destroying the cell.