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

\