Virology

• What are pathogens?

  infectious agents that cause disease in their hosts 
• Consequences of viruses?

  epidemics 
• Friedrich Loeffler + Paul Frosch & modern concept of a virus :  

  • Discovered first animal virus (foot and mouth disease) 
  • Closest to the modern concept of a virus: 
  • Filterable particle too small to be observed in light microscope but able to cause disease by multiplying in living cells 

   
• Development of tissue culture cell lines : 

  • Used to grow viruses in lab by infecting them with viruses 
  • Viewed under light microscope  
  • Led to advancements in research 

   
• What are different types of virology research (current)? : 

  • Virus diversity : - shape + size (more diverse than bacteria, plants + animals combined)
  • Virus epidemiology  
  • Viral pathogenesis 
  • Viral replication/interaction with host cells 
  • Viral particle structure 
  • Anti-viral 
  • Vaccines 
  • Gene therapy 
• What are viruses?

  • infectious, obligate intracellular parasites comprising genetic material surrounded by a protein coat

  
  

  • uses host cell machinery to replicate viral genome + produce viral proteins
  • assembles from proteins + genomes formed in infected cells
  • DON'T REPLICATE BY DIVISION
• Where do viruses exist?

  1. Outside host cells = inactive, possess few enzymes + can't reproduce outside living cells
  2. Inside host cells = as nucleic acids which transform host cells into virus factories
• Virus particles

  • viral genome surrounded by a capsid
  • capsid = protein coat (produces genetic material + aids transfer to host cells)
  • some capsids are surrounded by an envelope (lipid bilayer derived from host)
  • nucleocapsid = capsid + viral nucleic acid
  • virion = complete infectious virus particle
• Capsid structure

  • self assembles from multiple copies of structural proteins
  • individual protein subunit of a capsid = promoter
  • can be helical, icosahedral or complex
• Helical capsid

  • hollow tubes w/ protein walls
  • rigid/flexible
  • size of capsids = function of nucleic acids
  • helical array of promoters
  • viral nucleic acid spirals on the inside
• Icosahedral capsid

  • 20 triangular faces arranged around the surface of a sphere
  • most efficient way to enclose a space
  • assembles from pentamers or hexamer
  • assembled virus capsid = 12 pentamers
• Viral envelopes

  • lipo-protein bilayer derived from host
  • obtained when virus particles 'bud' through host cell membranes (plasma, or sometimes golgi/nuclear)
  • virus proteins embedded (spikes) : viral attachment to host antigenic site via spike proteins on virus surface + receptor on host cell surface
• Virion enzymes

  • variety of virions have enzymes
  • mostly within capsid
  • involved in nucleic acid replication
  • e.g. retrovirus (HIV) , reverse transcriptase (converts viral RNA into DNA), intergrase (integrates viral DNA into host genome)
• Central dogma of molecular biology (Francis Crick)

  • describes flow of genetic info in cells
  • (1) genetic info stored in the form of DNA
  • (2) DNA replicates
  • (3) DNA transcribed into RNA
  • (4) RNA translated into proteins via ribosome
  • not all viruses follow this as some are made of RNA and not DNA
• Viral genome

  • DNA or RNA
  • ss or ds
  • ss: '+' or '-' sense
  • virus mRNA = '+'
  • if viral genome of DNA/RNA is complementary to genome of mRNA = '+' , then reverse of mRNA genome = '-'
  • linear or circular
  • continuous or segmented
• Viral replication cycle

  • mechanisms depend on virion structure + genome
  • (1) attachment to host cell via specific receptors
  • (2) entry into host cell
  • (3) uncoating
  • (4) synthesis of viral nucleic acids + proteins
  • (5) self-assembly of nucleocaspids
  • (6) release from host cell
• non-enveloped virus replication cycle

  1. attachment via specific receptors
  2. entry via endocytosis
  3. uncoating
  4. macromolecular synthesis: synthesis of viral proteins and replication of viral genome
  5. assembly of viral particle (viral genome + proteins)
  6. release by lysis
• enveloped virus replication cycle

  1. attachment via specific receptors
  2. entry via endocytosis/membrane-fusion
  3. uncoating
  4. macromolecular synthesis: synthesis of viral proteins and replication of viral genome
  5. assembly of viral particles (viral genome + proteins)
  6. release by budding
• What does virus genome dictate?

  how a viral mRNA is synthesised and how the viral genome is replicated
• Baltimore classification - simplifies viral life cycles into 7 basic types, based on:

  • DNA or RNA
  • ds or ss (+ or -)
  • RT or not
  • (1) DNA viruses : ds DNA or ss DNA
  • (2) RNA viruses : ds RNA or ss RNA (+) or ss RNA (-)
  • (3) retro-transcribing viruses : ds DNA (RT) , ss RNA (RT)
  • every viral genome needs to be converted into mRNA to enable translation into proteins by host ribosomes
• Baltimore classification (2)

  • ds DNA = majority of DNA viruses
  • ss DNA = converted into ds
  • ds, ss (+), ss (-) RNA all require viral RdRP
  • ss (+) RNA = genome can be directly translated upon entry
  • ss (-) RNA = needs to be converted into + while bringing RdRp into infected cell
  • retroviruses (ds DNA + ss RNA) = bring RT + integrase into infected cell. ds DNA integrates into host cell genome
• Viral classification taxonomy

  • goal = categorise viruses into single classification scheme, reflecting their evolutionary relationships
  • ICTV = international committee on taxonomy of viruses, responsible for classification into taxa + naming taxa
  • 15 taxonomical hierarchical ranks
  • most used = family, genus, species
• Techniques used to measure number of infectious viral particles?

  1. plaque assay
  2. end-point dilution assay (TCID50)
• Plaque assay

  • determines the virus tirte (conc. of infectious viral particles) in a sample
  • 1, prepare serial dilutions of virus - containing sample
  • 2, inoculation of susceptible cells in culture
  • 3, after virus attaches, cover cells with agar to restrict diffusion
  • 4, original infected cells release viral progeny, spreading to neighbouring cells
  • 5, each infectious viral particle produces a plaque (circular zone of infected cells
  • 6, virus titre of original sample calculated as plaque-forming units (PFU)/ml
• End-point dilution assay (TCID50)

  • determines dilution of a virus required to infect 50% of inoculated cell cultures
  • 1, prepare serial dilutions of virus - containing sample
  • 2, inoculation of susceptible cells in culture
  • 3, incubate
  • 4, observe w/ light microscope , score CPE (cytopathic effect), CPE present (circular) = virus present and if CPE absent (sausage) = no virus
  • 5, virus of titre of original sample = TCID50 units/ml
  • end-point = dilution where 50% of cells are infected or display CPE
• Techniques used to measure number of viral particles:

  1. Electron microscopy : see viral particles + count them
  2. ELISA (enzyme-linked immunosorbent assay) : quantify viral proteins
  3. qPCR - quantify viral genome
• One step growth cycle

  • studies viral replication cycle
  • used to analyse virus + host factors that facilitate viral replication
  • 1, short exposure to high number of virions
  • 2, wash away virions that don't attach to cells
  • most cells infect at the same time
  • synchronous viral replication in most of the cells

  3 phases : eclipse, burst, plateau
• Routes of viral entry to humans:

  1. conjuctiva
  2. respiratory tract
  3. gastro-intestinal tract
  4. urogenital tract
  5. skin
  6. placenta
• Viral pathogenesis (1) : DISEASE

  • due to virus causing:
  • cell destruction
  • cellular dysfunction
  • immunosuppression
• Viral pathogenesis (2) : INFECTION

  • acute virus infection can be:
  • clearance : recovery
  • fatal : death
  • persistent : death or latent
  • can lead to:
  • cell/tissue tropism
  • subversion of host molecules/mechanisms
  • evasion from host responses
  • mutation
  • host can gain:
  • susceptibility, immunity, genetic variations in specific genes
• Tissue tropism: spectrum of tissues infected by a virus

  • ranges from limited to pantrophic (affects many types of cells)
  • determined by:
  • susceptibility, permissivity, accessibility + defense
• Effect of viral infection on cells

  • cell death : most enveloped viruses
  • cell dysfunction (cell transformation)
  • no effect
  • some viruses induce an alteration of host cell functions (pathological consequences)
  • transformation of host cells by viruses can lead to oncogenesis (cancer)
• Immune response to viral infections

  • host defences:
  • physical barriers: skin, mucus, saliva, stomach acid, tears
  • intrinsic : autophagy, apoptosis
  • innate : killer T-cells, dendritic cells, cytokines, complement
  • adaptive: T-lymphocytes, B-lymphocytes, antibodies
• Intrinsic + innate responses to viruses, Virus detection = immediate + not antigen specific

  • indirect: alteration of normal cellular processes
  • apoptosis = programmed cell death (blocks virus replication)
  • tissue - resident sentinel cell takes up apoptotic bodies
  • autophagy = membrane encloses virus + fuse w lysosome to degrade viral particles (phagophore -> autophagosome -> lysosome -> degradation)
• Intrinsic + innate responses, direct virus detection

  • PRRs (pattern recognition receptors)
  • 1, recognise viral nucleic acids + proteins
  • 2, signalling cascade
  • 3, activation of transcription factors IRF3/7 + NF/KB
  • 4, gene expression of:
  • inflammatory cytokines: soluble proteins that AFFECT OTHER CELLS BEHAVIOUR, secreted -> inflammation -> recruitment/activation of immune cells = (DCs instruct adaptive immunity + NK cells kill infected cells)
  • Type 1 IFN : cytokines that ACTIVATES ANTIVIRAL PROGRAMMES, secreted -> binds IFN receptor -> signalling cascade -> ISGs expression -> antiviral effect
• Intrinsic + innate responses, sentinel cells

  • dendritic cells (DCs), macrophages, natural killer cells (NK)
  • patrol tissues looking for signs of change
• Main mechanisms to prevent viral evasion:

  1. neutralising antibodies = adaptive immune response that provides long-lasting protection by blocking viral entry + promoting clearance
  2. cytotoxic T-lymphocytes = adaptive immune response that provides long-lasting protection by killing infected cells
  3. cell intrinsic = induced by type 1 IFNs that inhibit viral replication
  4. cytotoxic NK cells = directly kill infected cells
• Difference between innate + adaptive immune response

  • innate = not antigen-specific
  • adaptive = antigen-specific and long-lasting
• What happens when you don't clear viral infections?

  • IFN (inflammatory response)
  • needs to be controlled
  • Locally : many ISGs (interferon-stimulated gene) inhibit viral replication but damaging to tissues, toxic for cells 
  • In circulation: too much ISGs secreted locally -> enter bloodstream -> trigger flu-like symptoms (fever, chills, nausea, malaise) 
  • There are regulators of IFN signaling / inflammatory cytokines to avoid pathology once viral replication is controlled 
• Immunopathology : 

  • due to: 
  • Uncontrolled innate immune response (IFN/Inflammatory cytokines)  
  • Damaging effects of adaptive immune response 
  •  symptoms of viral disease (fever, tissue damage, aches, pains, nausea)  
  • mainly a consequence of host response to infection.