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
  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
  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,
  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
    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