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Topic 12: Viruses

  • Viruses - acellular infectious agents
  • Ebola Virus
    • first identified in 1976 (2 outbreak near Ebola river)
    • Genus Ebolavirus - total 5 species, 4 species pathogenic to human, Reston virus affects primates only
    • RNA, attach to cell surface, fruit bat is the suspected host
    • 25-90% fatality, 4-10 day incubation period
    • direct contact transmission
    • 3 months after recovery, found in semen - sexual transmission
    • burial rituals cause infection
    • dog can be infected, antibodies detected, no symptom
    • macrophages, dendritic cells, and monocytes are the target
    • PCR base test for clinical diagnosis
  • Biosafety Levels (BSL)
    • BSL - 1
    • micro lab, minimal potential threat, no pathogen, standard open lab benches without the use of special equipment
    • BSL - 2
    • pathogen, moderate hazard, personal protection needed, need to be immunized for Hep B, TB test, immunocompromised/immunosuppressed possibly denied, ex: Hep A, B, C, HIV, flu, MRSA
    • BSL - 3
    • self-closing door, separate entrance required, registration with government required, lab coats need to be dontaminated before laundry, ex: west nile virus, anthrax, rabies virus, SARS virus
    • BSL - 4
    • lift-threatening diseases, separate building, pospressure air supplied, full body suit, ex: ebola
  • Enzootic vs Epizootic
    • Enzootic
    • endemic within
    • Epizootic
    • epidemic among animals
  • Discovery of Viruses
    • Charles Chamberland (1884)
    • developed porcelain bacterial filters used later in discovery of viruses
    • Dimitri Ivanowski (1892)
    • demonstrated that causative agent of tobacco mosiac disease passed through bacterial filters
    • thought agent was toxin
    • Martinus Beikerinck (1898-1900)
    • showed that causative agent of tobacco mosaic disease was still infectious after filtration
    • referred to agent as filterable virus
    • Wendell Stanley (1935)
    • discovered that viruses were made of nucleic acid and protein
  • Virus Characteristics
    • exceptionally small
    • contain a single type of nucleic acid, either DNA or RNA
    • possesses a protein coat surrounding the nucleic acid (may have an envelope outside of the protein coat) = virion
    • multiply inside living cells using the metabolic machinery of the cell
    • an exceptionally complex aggregation of nonliving chemicals
    • viruses have no metabolic machinery of their own and are, therefore, obligate “parasites”
    • comparison with prokaryotes (include bacteria)
    • can evolve & multiply
    • cannot consist of cell, metabolize, or respond to stimuli
    • comparison with bacteria only
    • they can pass through bacteriological filters and sensitive to interferon
    • do not have plasma membrane, cannot reproduce through binary fission, do not possess both DNA and RNA, no ATP-generating metabolism, no ribosomes, and not sensitive to antibiotics
  • The structure of viruses
    • cannot reproduce independently of living cells nor carry out cell division as in prokaryotes and eukaryotes
    • size range is ~10-800 nm in diameter with most viruses too small to be seens with the light microscope
    • all virions contain a nucelocapsid which is composed of nucleic acid (DNA or RNA) and a protein coat (capsid)
    • some viruses consist only of a nucleocapsid, others have additional components
    • envelopes
    • virions having envelopes = enveloped viruses
    • virions lacking envelopes - naked viruses
  • Generalized Structure of Viruses
    • naked virus
    • capsid and nucleic acid
    • enveloped virus
    • capsid, nucleic acid, envelope, and spike
    • range from ~ 10 to 800 nm
  • Virus Structure
    • capsid: protein coat surrounding the nucleic acid (DNA or RNA)
    • composed of protein subunits called capsomeres
    • protein molecules may be the same or different
    • capsomeres can be arranged in several configurations
  • Virus Morphology
    • classified into four major groups
    • helical viruses
      • resemble long rods and may be flexible or rigid; the capsid is helical surrounding the nucleic acid
      • tobacco mosaic virus (discovered from tobacco plants)
      • (+) sense ssRNA - single strand RNA → ready to infect
    • polyhedral viruses
      • a many-sided virus particle; the capsid is usually in the shape of an icosahedron with ~20 (min 12 triangular faces and 12 corners)
      • adeno dsDNA
      • Rhino ss +RNA - single strand RNA → ready to infect???
    • enveloped viruses
      • ex: covid
      • has envelope and spike
      • spike attaches to host and is specific (lock and key method)
      • envelope: surrounds the capsid in some viruses
        • consists of some combination of lipids, proteins, and carbohydrates
        • may be synthesized during virus production or be a part of the plasma membrane of the host cell
      • +sense ssRNA
    • complex viruses
      • new antibiotics? - use of bacteriophage targeting
  • Viruses with Capsids of Complex Symmetry
    • special types of icosahedral shape = prolate, variation of icosahedral shape in phage
    • many viruses do not fit into the category of having helical or icosahedral capsids
    • examples are the poxviruses and large bacteriophages
  • Virion Enzymes
    • it was first erroneously thought that all virions lacked enzymes
    • now known a variety of virions have enzymes
    • some are associated with the envelope or capsid but most are within the capsid
  • Viral Envelopes and Enzymes
    • many viruses are bound by an outer, flexible, membranous layer called the envelope - for invasion
    • in eukaryotic viruses, some envelope are proteins, which are viral encoded, may project from the envelope surface as spikes or peplomers
    • host specificity/infectivity
  • Virus Structure
    • Nucleic acid - linear, circular or segmented
    • DNA - doubled stranded or single stranded (herpes, chickenpox)
    • RNA - double stranded or single stranded (ebola, flu, west nile)
    • RNA > DNA virus
    • RNA virus (aka ribovirus, excludes retrovirus)
    • Positive sense strand - same as mRNA, ready for translation → protein synthesis
    • Negative sense strand - complementary to mRNA
    • virion’s enzyme RNA-dependent RNA polymerase
    • transcriptase - ready for translation → protein synthesis
    • retrovirus - RNA, DNA, RNA (HIV) - target for drug
  • Retrovirus
    • “The central dogma of molecular biology deals with the detailed residue-by-residue \n transfer of sequential information. It states that such information cannot be transferred back from protein to either protein or nucleic acid.” —Francis Crick
    • DNA makes RNA and RNA makes protein
    • “retro” comes from reversal
    • Retrotransposons in all eukaryotes
  • Viral Mutation
    • Antigenic drift: point mutation, mostly “silent”
    • Antigenic shift: major genome change due to recombination, last year’s flu
    • RNA virus has higher mutation rate than DNA virus
  • The Cultivation of Viruses
    • requires inoculation of appropriate living host
    • Bacteriophages can be grown in bacterial cultures using the plaque method
    • Animal viruses must be grown in cell culture or embryonated eggs
  • Hosts for animal viruses
    • Embryonated (fertilized) eggs
    • tissue (cell) cultures
    • monolayers of animal cells
    • plaques
      • localized area of cellular destruction and lysis
    • cytopathic effects
    • microscopic or macroscopic degenerative changes or abnormalities in host cells and tissues
  • How to make Flu vaccine
    • WHO “predict” flu strains
    • 1 virus/egg = 1 dose
    • Normally 3-4 strains
    • Need ____ doses x 3 –4 eggs
    • *130 million in 2013 = ½ million hens (produce 250 eggs/year)
    • Spin to collect serum, chemically “kill” virus
  • Cell-Based Flu vaccine (approved 2012)
    • use mammalian cell line, a bit faster, do not need to rely on egg supply
    • Recombinant Flu vaccine (approved 2013)
    • flu protein (immune response inducing) combined with another virus, add to insect cell, faster
    • Flu vaccine contains thimerosal (organomercury, aka merthiolate) = bacteriostatic, thus not needed in a single dose shot/mist
    • Tattoo inks, skin test allergens, vaccines (removed from children’s vaccines –autism link?)
  • Measuring concentration of infectious units
    • plaque assays
    • dilutions of virus preparation made and plated on lawn of host cells
    • number of plaques counted
    • results expressed as plaque-forming units (PFU)
    • Assumes each PFU was the result of the infection of a bacterium by one virus particle which then radiated through lysis of infected bacteria
  • Classifcation of Bacterial and Archaeal Viruses
    • the International Committee for the Taxonomy of Viruses (ICTV) standardizes the viral classification
    • ~2,300 viruses have been classified, most being viruses of eukaryotes and bacteria (some 5000)
    • ~40 Archaeal viruses have been identified; ~ 15 of these have been assigned to virus taxa
    • based on two major criteria
    • capsid structure (but now that is being questioned)
    • nucleic acid properties
  • Taxonomic Classification
    • Order (-virales) Family (-viridae) Subfamily(-virinae) Genus (-virus) Species(-virus)
    • In the current (2011) ICTV taxonomy, six orders have been established, the Caudovirales, Herpesvirales, Mononegavirales, Nidovirales, Picornavirales and Tymovirales. A seventh order \n Ligamenvirales has also been proposed.
    • In total there are 6 orders, 87 families, 19 subfamilies, 349 genera, about 2,284 species and over 3,000 types yet unclassified
    • **Based primarily on structural components
  • David Baltimore
    • First to describe RNA dependent RNA polymerase----which virus? During his PhD work
    • Early faculty years at MIT discovered reverse transcriptase, discovered retrovirus
    • Nobel prize in 1975
    • Developed Baltimore classification via viral replication method
  • Baltimore Classification
    • I: dsDNA viruses (e.g. Adenoviruses, Herpesviruses, \n Poxviruses)
    • II: ssDNA viruses (+ strand or "sense") DNA (e.g. Parvoviruses)
    • III: dsRNA viruses (e.g. Reoviruses)
    • IV: (+)ssRNA viruses (+ strand or sense) RNA (e.g. Picornaviruses, Togaviruses)
    • V: (−)ssRNA viruses (− strand or antisense) RNA (e.g. Orthomyxoviruses = flu, Rhabdoviruses)
    • VI: ssRNA-RT viruses (+ strand or sense) RNA with DNA intermediate in life-cycle (e.g. Retroviruses)
    • VII: dsDNA-RT viruses (e.g. Hepadnaviruses, Hep B)
  • Absorption and Penetration
    • receptor sites
    • specific surface structures on host to which viruses attach
    • specific for each virus; can be proteins, lipopolysaccharides, techoic acids, etc.
  • Life Cycle of dsDNA T4 Phage of E. coli
    • Adsorption to specific receptor site – porin protein and LPS
    • Penetration of the cell wall – peptidoglycan degrades
    • Insertion of the viral nucleic acid into the host cell
    • Transcription → early mRNA
    • Translation of early mRNA resulting in production of protein factors and enzymes involved in phage DNA synthesis
    • Transcription →late mRNA
    • Translation of late mRNA resulting in synthesis of capsid proteins, proteins required for phage assembly and proteins required for cell lysis and phage release
    • Cell lysis and phage release – 12 minutes after initial absorption (100-150 new phages)
  • Synthesis of Phage Nucleic Acids and Proteins
    • most ds DNA viruses
    • use their DNA genome as a template for mRNA synthesis
      • the mRNA is translated to produce viral proteins
  • The T4 Genome
    • a large proportion of the genome codes for replication-related products including
    • protein subunits of its replisome
    • enzymes needed for DNA synthesis
      • some of these enzymes synthesize hydroxymethylcytosine (HMC), a modified nucleotide that replaces cytosine in T4 DNA
      • Intron present
  • Synthesis of T4 DNA
    • contains hydroxymethyl-cytosine (HMC) instead of cytosine
    • synthesized by two phage encoded enzymes
    • then HMC glucosylated protects phage DNA from the host restriction endonucleases so that new phage nucleic acids cannot be damaged during their synthesis enzymes that cleave DNA at specific sequences
  • Assembly of Phage Particles
    • complex self-assembly process
    • involves viral proteins as well as some host cell factors
  • Release of Phage Particles
    • in T4 - E. coli system, ~150 viral particles are released
    • two proteins are involved in process
      • T4 lysozyme attacks the E. coli cell wall
      • holin creates holes in the E. coli plasma membrane
  • Reproduction of RNA Phages
    • most are positive sense RNA viruses
    • incoming RNA acts as mRNA and directs the synthesis of phage proteins
    • double-stranded RNA viruses have also been discovered
  • Temperate Bacteriophages and Lysogeny
    • temperate phages have two reproductive options
    • reproduce lytically as virulent phages do
    • remain within host cell without destroying it
      • done by many temperate phages by integration of their genome with the host genome in a relationship called lysogeny
  • Lysogeny
    • prophage
    • integrated phage genome
    • lysogens (lysogenic bacteria)
    • infected bacterial host
    • temperate phages
      • phages able to establish lysogeny
  • Distinctive characteristics of Lysogenic Bacteria
    • they are immune to superinfection (ex. Once Lambdainfected, no secondary Lambdasecondary infection)
    • under appropriate conditions they will lyse and release phage particles
    • this occurs when conditions in the cell cause the prophage to initiate synthesis of new \n phage particles, a process called induction
  • Lysogenic conversion
    • change in host phenotype induced by lysogeny
    • e.g., modification of Salmonella LPS structure
    • e.g., production of diphtheria toxin by Corynebacterium diphtheriae
  • Prion
    • BSE (Bovine Spongeform Encephalopathy)
    • CJD (Creutzfeldt-Jakob Disease)
  • Function of Prion
    • In human, chaperons are located in ER
    • Physiologically unknown-possibly related to myelin repair in Shwann cell
    • 2005 long-term memory retention indicated
    • 2006 self-renewal of stem cell in bone marrow indicated
  • Protein replication
    • Process not fully understood in protein only replication
    • Heterodimer model and fibril model
  • Human Diseases caused by Prions
    • Creutzfeldt-Jakob Disease
    • Iatrogenic – prion-contaminated human growth hormone, dura mattaer graft
    • New variant – infection from Bovine prions??
    • Familial – Germ-line mutation in the PrP gene
    • Sporadic – Somatic mutation or spontaneous conversion into disease form??
    • Kuru
    • Infection through ritualistic cannibalism in New Guinea
  • FDA and USDA standards
    • Test Bovine feed for Ruminant feed contamination (after 2009)
    • “Downers” examined by USDA vet, need clearance in order to be processed for human consumption
    • CNS and spinal fluid contamination ban via air gun slaughter