Virology: Virus Structure, Infection & Nomenclature

Viruses

• Filterable agents that represent a debatable form of life.
• They are obligate intracellular agents, requiring a host to replicate.
• The viral genome is covered in protein, replicating via assembly, and can be either DNA or RNA.
• Viruses possess a protein capsid which can be icosahedral, featuring triangle-shaped plates, or helical, surrounding the genome.
• Some viruses have a lipid membrane or envelope, but not all.
• Nucleic acid types in viruses can be either DNA or RNA; double or single-stranded; linear or circular; continuous or segmented.
• Single-stranded RNA viruses can have positive (+) or negative (-) polarity.
• Retroviruses, like HIV, use (+) RNA to create DNA.

Virus Nomenclature

• Order names end in "virales," and family names end in "viridae."
• Sub-family names end in "virinae," followed by genus and species.

Steps for Infection

• Adsorption: The virus protein attaches to the cell, depending on the host range and which species the virus can infect.
• Tissue Tropism: Determines which tissue of a specific species the virus can infect.
• Penetration: The virus travels across a cell membrane via endocytosis.
• Uncoating: Typically occurs at the same time as penetration, releasing the genome into the cell after the capsid breaks down.

Synthesis

• Genome Replication:

  • DNA replication happens inside the nucleus and uses the cell's mechanisms, exception is Poxviruses bringing their own polymerase
  • RNA replication occurs inside the cytoplasm and depends on RNA-Dependent RNA Polymerases (RdRp), often brought in with the virus, using cells transcription machinery, with an exception being retroviruses which transform RNA to DNA upon entry

• Assembly happens spontaneously or through cell-mediation, generally at the site of synthesis.

Release

• Naked (Non-Enveloped) Viruses: Cause "necrotic," lysis/metabolic death, killing the cell and leading to a loss of cell membrane integrity.
• Enveloped Viruses: Induce "apoptotic" cell death through budding of the plasma membrane or other interior membranes, are more stable, leading to slower than necrotic death, and cause programmed cell death where the cell shrinks and kills itself.

Viral Infections

• Acute/Lytic Infections: Are resolved by the immune system, are of short duration, and are tissue-restricted
• Persistent infections: Cause late complications after the acute infection, and are slow/long infections
  • Latent Infections: Persistent but undetectable
  • Chronic Infections: Persistent and detectable
• Retroviruses: Have a complex capsid and special enzymes
  • Reverse Transcriptase: Turns RNA into DNA
  • Integrase: Allows DNA to enter the nucleus as provirus DNA or viral DNA

Cancer and Viruses

• Up to 20% of cancers may be associated with viral infections.
• Epstein-Barr virus (EBV): Can cause mono and is associated with Hodgkin's lymphoma.
• Oncogenes: Cancer genes that are viruses and the cell cycle
  • Can stimulate or prevent the cell cycle, stopping tumor suppressing genes

Cell Cultures

• Viruses need living cells to grow.
• Living animals, mice, embryonated eggs, or tissues grown in petri dishes are used in cell cultures. Adherence involves attachment to a plate
• Cytopathic effect: Changes occur with infection and cell loss.
  • Vacuoles/bubbles occur.
  • There is cell fusion and inclusion bodies. Virus-Like Agents
• Vacuoles/bubbles occur.
• There is cell fusion and inclusion bodies.
• Neighboring cells are fused with infected cells due to the outer proteins of the virus attaching to other cells' receptors and occur in the eveloped viruses

Viral Quantification Techniques

• Plaque Assay: Counting holes in the plate, called plaques, which are sites of cell death, to count the virion, viable or infectious viral particles.
• Hemagglutination Assay: Uses viruses capable of clumping red blood cells (influenza, measles, adenoviruses).
• Measured by Titer: Highest dilution where clumping occurred and dots in the liquid represent non-clumping
• PCR: DNA process that uses polymerase chain reaction to amplify a region of the viral genome until detectable.
• RNA PCR: Uses reverse transcriptase from retroviruses that transforms RNA into DNA for use in a regular PCR.

Virus-Like Agents

• Viroids: Plant pathogens that consist of naked RNA, have no membrane, and cannot break through anything.
• They enter through broken plant membrane
• A molecular parasite
• Satellite Virus: Cannot replicate without a "helper" virus and cannot transmit by themselves.

Prions

• They are most significant agents derived from a normal protein, misfolded by mutation.
• They are stable but neurotoxic, causing spongiform encephalopathy
• They can transmit as a template for other host proteins
• They are not killed by normal sterilization methods, are stronger than endospores, and are resistant to both ionizing and non-ionizing radiation. They are killed by strong bleach or by autoclaving

Bacterial Viruses (Bacteriophages)

• To infect, they have to get through a cell wall
• They punch through layers using the lysozyme and injecting the viral DNA
• Lytic Replication: Replicates and kills; forms clear holes in the bacterial lawn on a lytic plaque assay where diffusion is limited by the agar matrix
• Temperate: Causes lysogenic conversion when phage DNA enters bacterial DNA as "Prophage" DNA
  • Bacteria are now called a lysogen with a new phenotype, which can produce toxins
  • Some diseases are caused by toxin-producing phages
    • Most phage species are temperate
    • Clear spots in thebacterial warn is displayed by a temperate palque assay

Lambda Replication

• Infects E. Coli
• This is when one can lyse or become lysogenic
• A lysogen may lyse when encountering cell damage or stress
  • Triggers phage replication followed by lysis

Bacterial Genetics

• Experiments helped prove that DNA inherited in Large numbers of phages in stool
• Hershey Chase used Radioactive phages.
  • Allows tracing of transduction pieces
  • Sulfur: Causes radioactive protein shell
  • Phosphorus: Causes radioactive DNA
    • Only DNA enters the cell. The Phage shell does NOT
• Griffith and Avery used 2 strains of bacteria
  • One with a capsule, smooth. One without a capsule, rough. - Capsules are pathogenicity factors -Dead smooth strain alone: Host lives
    • Living rough strain: Host lives
    • Dead smooth and living rough strain together: Dead host
    • Capsule expression gene was transferred
    • Caused a new phenotype

DNA

• Deoxyribonucleic Acid: Composed of nucleotides
  • Structure:
    • Phosphate - Binds to sugar of other nucleotides
    • Sugar - Backbone of DNA
    • Nitrogenous base
      • Purine: Adenine, Guanine
      • Pyrimidines: Thymine, Cytosine
  • Paired to form a double helix:
    • Pairing
      • Adenine-Thymine
      • Guanine-Cytosine
      • Changing the chemical structure can alter pairing
      • Guanine is altered to bond to thymine
      • Replicates as adenine

Replication

• Is semi-conservative with 1 original strand and 1 new strand
• Enzyme Catalyzed:
  • Helicase: Unzips DNA
  • Primase: Adds RNA as a primer at the origin strand of nucleotides
  • DNA Polymerase III: Adds DNA chain
  • DNA Polymerase I: Removes RNA primer and is replaced with DNA
  • Ligase: Finalizes binding of nucleotides together
  • Topoisomerases: Untangles DNA temporarily breaks and rejoins strands, and controls coiling

Transcription

• DNA copied to RNA by RNA Polymerases

Polymerases

• DNA: Begins at the origin and continues until the end of the strain.
• RNA: Begins at a promoter region, transcribes genome, needs no primer, and continues until the termination sequence of the gene.

Modification of mRNA

• Occurs in eukaryotes and features a 5' cap, required to interact with ribosomes - The nuclear envelope separates RNA synthesis from protein synthesis

      - Features a 3' tail that promotes stability and structure
  

  • Removal of interon(s) of Splicing: Certain parts of RNA that allow for customization

Prokaryotes

• There is no splicing, no interons, no modification and no separation of RNA and protein synthesis regarding the genetic code which is 64 combinations, 20 amino acids, 2-4 combinations per amino acid for start codon AUG: - Methionine. N-Formyl Methionine in Bacteria can be removed from sequence later

Genetic Variation

• Sex is the Accumulation of new genetic material
• Mutations:
  • Point Specific
    • Silent: No amino acid change, not significant
      • Mis-Sense: Amino acid changed, can be significant, and protein is changed that can lead to function change
    • Non-Sense: Amino acid changed to STOP codon, very significant, and loss of protein
• Addition/Deletion: Frameshift with addition or deletion of a base scrambles protein that shifts the reading frames, and protein function is lost

Mechanisms of Mutations

• Spontaneous:

  • Polymerase error rate
  • DNA Polymerase Proofreading
    • Detects and corrects mutations
    • Not perfect
  • Low rate of mutation

• Induced:

  • Physical/Chemical:
    • Hydrogen bonding disruption
    • Nucleotide modification
    • High rate of mutation

• Other Mutagens:

  • Base Analogs:
    • Non-reactive
    • Already altered nucleotides
    • Causes incorrect pairing
  • Intercalating Agent:
    • Adds an extra base
    • Distorts DNA structure -Induces a frameshift mutation
  • Radiation:
    • Needs to be at relatively high levels
      • Non-ionizing:
      • Ultraviolet (UV)
        • Ionizing:
          • X-Rays
          • Gamma Rays
          • Knocks electrons out of place
          • Causes free radicals
          • Damages DNA Polymerase
          • more error prone

Ultraviolet Radiation

• Ultraviolet (UV): Decreased wavelength is more dangerous
  • UVC: Most harmful, lowest wavelength, blocked by ozone
  • UVB: Less dangerous than UVC
  • UVA: Least dangerous, highest wavelength, most common
  • Thymine Dimerization: Covalent bonding of thymine due to ultraviolet radiation
  • UVB and UVC are more common to cause

Repair Mechanisms

• Light Repair:
  • Photoreactivation
  • Enzyme targets light energy
  • Focuses at incorrect bond
• Dark Repair:
  • Excision
  • Cuts out damaged nucleotides
  • Replaced/repaired
    • DNA Polymerase I
    • Ligase
• SOS Repair:
  • Last resort
  • Polymerase reads through the mutation
  • no proofreading can occur
  • Ionizing Radiation: Damages DNA and Polymerase

Testing for Mutagens

• Ames Assay:
  • Bacteria used to test - Typically mutated salmonella - Only grows with histadine -Reversion mutation: Undoes a previous mutation Salmonella can then grow without histadine

Horizontal Gene Transfer

• Between cells: Transformation Donating cell dies Receiving cell picks up dead cells DNA Some cells more susceptible to uptake - Called "Competent" cells - Plasmids replicate before being attacked Transduction - Donor cell dies - Phage-Mediated Some phages from lysogens have host DNA instead of phage - Injected into another host cell

      - F plasmid/factor transmitted
       Donors F plasmid replicates prior to transfer
       Recipient goes from F- to F+
  

Within Cell

   - Transposition:  -    Transposons Segmented, catalyzed by transposases  

How to know if a trait has been acquired?

       Does the new phenotype function differently from the parent?   - If yes = Acquired  - If no = Not Acquired

DNA Sequencing

     Helps prove that bacterial genetics is inherited
- Methods are needed to determine what traits are aquired

• Sanger:
  -Uses individual small segments of DNA
• High Thoughput: Uses a large number of DNA segments
• Sequence Maps: Small segments of DNA stitched together - Reveals the whole gene
• Databases: Computerized data files with the identities of already sequenced stretches of DNA

DNA Fingerprinting/Profiling

     -Approximation of genetic identity by specific gene cleavage
      - Restriction Endonucleases:  Bacterial enzymes, cleaves DNA at different sequences
      -

Terminology of sterilization

-Sterilization: - Complete destruction of all microorganisms including endospores - Disinfection Reduces viable pathogen numbers and Reduces viable pathogen numbers on living tissue E.g. alcohol or CHG -Pasteurization: Reduces food spoilage and number of pathogens -Sanitization: Removes microbe populations and scrubbing with soap is a way to achieve this -Preservation: Preservation needs risk to individuals

Considerations

• Risk to individuals: - Critical: Sterilization required & Direct contact with the vascular system - Semi-critical: Typically sterilization & Direct contact with the vascular system - Non-critical: Sterilization not required & Type of threat : Direct contact with intact skin

Type of threat

• Resistance: -Highest: - Most resistant, Endospoers, tuberculosis has a waxy layer - Moderate -Pseudomonas, Aureus, Protozoa, bacteria, Is the number of organisms or the degree of threat - Fungi

What to use to kill specific items

• Microbial Death -Not instantaneous

     - Radiation Radiation / Energy Transfer
  

  • Non-lonizing light rays, can kill rays

Chemical control

  -Chemical Control is very important to not risk any illness or further infection

• Filtering: stop bacteria. -High Efficiency Particulate Air filters (HEPA) are needed - Chemical Germacides: Most disinfectants target proteins

  • High pH Temperature Organic matter

  • Liquid or gas