Viruses

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Viruses

• Microbiology 221

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Viruses

• Obligate intracellular parasites
• They are able to reproduce their life cycle only
  within the cell of their host
• They usually have an external capsid composed of
  proteins
• Inner core of nucleic acid( dsDNA, ssDNA,dsRNA,
  and ssRNA)
• Specificity for the host

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Classification of viruses

• According to Baltimore classification, viruses
  are divided into the following seven classes
• dsDNA viruses
• ssDNA viruses 
• dsRNA viruses 
• ()-sense ssRNA viruses 
• (-)-sense ssRNA viruses 
• RNA reverse transcribing viruses
• DNA reverse transcribing viruses
• where "ds" represents "double strand" and "ss"
  denotes "single strand".  

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Table of Viruses
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Viral Capsids
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Capsids

• Capsids are made from protein subunits called
  capsomeres
• In some viruses, the capsomeres are all the same
  Geometric shape in others the sub units vary

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Antigenic spikes

• Some viruses have molecules inserted into the
  outer covering of the virus
• These may be glycoproteins
• In some cases these serve as a means of attaching
  to the host cell
• They are specific for one cell type

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Capsid Shapes
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Ebola Shepherds Crook
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Enveloped Virus
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Envelopes

• Lipid composition
• Acquired from the host when the virus exits the
  cell
• Provides a means for viruses to elude the immune
  system of the host by surrounding itself with the
  host envelope
• Enveloped viruses may be more vulnerable to
  chemical agents(chlorine, hydrogen peroxide, and
  phenol)
• They do not survive well on surfaces

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Viral Life Cycle Factors Influencing the Life
Cycle

• Nucleic acid
• Enveloped or naked
• Shape
• Host

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Host Range

• Primates
• Vertebrates ( birds)
• Plants
• Insects
• Bacteria

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Bacteriophages

• Viruses that infect bacterial cells
• Genome can be DNA or RNA
• Bind to specific receptors that are proteins or
  carbohydrates in the bacterial cell wall

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Bacteriophage structure
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Bacteriophages
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PhiX174 Spherical Bacteriophage

• Interesting to study because of its overlapping
  genes which is a model of efficiency

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T- 4 Bacteriophage

• Studied by Luria and Delbruck at Cold Spring
  Harbor
• Ds DNA virus
• 168, 800 base pairs
• Phage life cycles studied by Luria and Delbruck

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Filamentous phages

• Fd
• Filamentous
• Circular ss DNA
• Lies in the middle of the filament
• Infects through the pilus
• Create a symbiotic relationship with the host

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M 13

• Used for Genetic engineering experiments

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Bacteriophages Life Cycles

• Lytic They attach and enter the bacterial cell.
  Complete their life cycle by bursting the
  bacterial cell lysis
• Lysogenic They attach and enter the bacterial
  cell. The virus then integrates into the
  bacterial cell as a prophage

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Process of Infection

• Attachment
• Injection
• Hostile take over( lytic)
• Integration( lysogenic) Genetic control
• Early genes/late genes
• Replication of nucleic acid
• Production of viral proteins
• Assembly
• Lysis

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Lytic Life Cycle( Virulent)

• The viral genome contains a promoter that
  attaches to host cell RNA Polymerase
• Early genes code for those proteins that shut
  down the host, replicate nucleic acid, and code
  for vital proteins
• Nuclease genes, are capable of digesting host DNA
  so that the bases can be used for the production
  of new virions
• Late genes code for viral capsid and for those
  proteins that lead to lysis of the host cell

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Lytic Cycle

• Strict control
• Do not want to lyse the cell prior to the
  completion of assembly
• Usually only one virus in a cell at a time
• Recombination occurs between the two viruses if
  more than one is present

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Lysogenic Life Cycle

• Bacteriophages that do not lyse the bacteria cell
  are referred to as temperate
• Lysogenic bacteria contain a copy of the virus
  which is non infective and is known as the
  prophage
• The prophage can remain inactive through many
  cell division
• Bacteria can switch between lytic and lysogenic
  life styles
• When the host is stressed or damaged by mutagens,
  this stimulates the prophase to excise itself and

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Generalized Transduction

• Any part of bacterial genome can be transferred
• Occurs during lytic cycle
• During viral assembly, fragments of host DNA
  mistakenly packaged into phage head
• generalized transducing particle

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Generalized transduction
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Specialized transduction
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Lambda Phage

• Bacteriophage Lambda is a temperate phage meaning
  that it can undergo either a lytic or lysogenic
  cycle
• The phage regulates this cycle genetically
  through a switch

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Attachment

• Bacteriophage Lambda binds to the target E. coli
  cell, the tail tip binding to a maltose receptor.
  
• The linear phage genome is injected into the
  cell, and immediately circularises.
• Transcription starts
• There are two regulatory viral proteins, CI and
  Cro
• CI and Cro compete for the operator promoter
  sites on the phage DNA
• When the bacterial host is healthy CI accumulates
  and the lambda integrates into the bacterial
  genome and stays in this position
• .

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Control of lysogeny and lytic cycle

• Genes needed to establish lysogeny
• cI yes
• cII yes
• cIII yes

• Genes needed for maintenance of lysogeny
• cI yes
• cII no
• cIII no

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Insertion sequences
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Genetic Control of Lytic and Lysogenic Phage
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Lytic vs. Lysogenic

• When Cro is low, CI maintains the lysogenic life
  style
• When Cro accumulates due to damage of the host
  DNA or other unsuitable environmental conditions,
  the virus switches to the lytic life style
• This activates promoters for phage replication
  and protein synthesis
• Serves as a model for understanding viral
  infectivity and genetic control

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Integration

• Integration of bacteriophage lambda requires one
  phage-encoded protein - Int, which is the
  integrase - and one bacterial protein - IHF,
  which is Integration Host Factor.
• Both of these proteins bind to sites on the P and
  P' arms of attP to form a complex in which the
  central conserved 15 bp elements of attP and attB
  are properly aligned.
• The integrase enzyme carries out all of the steps
  of the recombination reaction, which includes a
  short 7 bp branch migration.

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Enzymes and Recombination

• The strand exchange reaction involves staggered
  cuts that are 6 to 8 bp apart within the
  recognition sequence.
• All of the strand cleavage and re-joining
  reactions proceed through a series of
  transesterification reactions like those mediated
  by type I topoisomerases.

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Excision of bacteriophages

• Excision of bacteriophage lambda requires two
  phage-encoded proteins
• Int (again!) and Xis, which is an excisionase. It
  also requires several bacterial proteins.
• In addition to IHF, a protein called Fis is
  required.
• All of these proteins bind to sites on the P and
  P' arms of attL and attR forming a complex in
  which the central conserved 15 bp elements of
  attL and attR are properly aligned to promote
  excision of the prophage.

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Lytic Cycle

• Lytic Lifestyle
• The 'late early' transcripts -genes for
  replication of the lambda genome.
• The lambda genome is replicated in preparation
  for daughter phage production.
• Transcription from the R' promoter can now extend
  to produce mRNA for the lysis and the structural
  proteins.
• Structural proteins and phage genomes self
  assemble into new phage particles.
• Lytic proteins build sufficiently far in
  concentration to cause cell lysis, and the mature
  phage particles escape.

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Bacteriophage growth curve
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Plaque Assay

• Eclipse Period- Penetration through biosynthesis
• Latent-Spans from penetration up to the point of
  phage release

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Lambda and Plaques

• The plaque produced by Lambda had a different
  appearance on the Petri Dish.
• It is considered to be turbid rather than clear
• The turbidiy is the result of the growth of phage
  immune lysogens in the plaque
• The agar surface contains a ratio of about a
  phage /107 bacteria

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Plaque assay
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Methodology

• Grow bacteria to log phase
• Prepare dilutions of bacteriophage
• Mix bacteria with viruses or overlay bacteria
  with suspension of viral particles
• Incubate
• Count infectious particles based upon the number
  of plaques
• Plaques are clear areas indicating lysis of
  infected cells

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Plaque Assay
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MOI

• Average number of phages /bacterium
• After several lytic cycles the MOI( multiplicity
  of infection) gets higher due to the release of
  phage particles

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Horizontal Transfer of Genes

• The transfer of genes or blocks of genes
• ( pathogenicity islands) from bacterium to
  bacterium or virus to bacteria or virus to virus
• Has resulted in many changes in microbes that
  have led to increase in pathogenicity and
  accumulation of virulence genes, not just
  resistance

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Streptococcus pyogenes

• There are 15 prophages that have been identified
  in E. coli
• These prophages belong to the group Siphoridae
• All but one of these produce a toxin
• In both strep and staph the prophage is found
  at the site of recombination

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Virulence and Streptococcus pyogenes

• Streptococcal pyrogenic exotoxins(SPE)
  contribute to the diverse symptoms of a
  streptococcal infection.
• These antigens compare to Staphylococcal
  antigens of the same type.
• The A C genes coding for these toxins were
  horizontally transferred from strain to strain by
  a lysogenic bacteriophage.
• In addition the genes contributed by the phages
  produce hyaluronidase, mitogenic factor, and
  leukocyte( WBC) toxins

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Pathogens with bacteriophages that cause
complications

• Corynebacterium diphtheriae
• Vibrio cholerae
• enterotoxogenic E. coli
• Staphylococcus aureus
• Clostridium botulinum
• Staphylococcus aureus and Streptococcus pyogenes
  Toxic shock syndrome

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Toxic shock
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Parvoviridae

• Viral family Parvoviridae
• single-stranded DNA naked polyhedral capsid
• Size 18-25nm
• Examples and diseases parvoviruses (roseola,
  fetal death, gastroenteritis some depend on
  coinfection with adenoviruses)

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Papovaviridae

• double-stranded, DNA naked polyhedral capsid
• Viral family Papovaviridae circular dsDNA
• Size 40-57nm
• Examples and diseases human papilloma viruses
  (HPV benign warts and genital warts genital and
  rectal cancers)

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Adenovirus

• Viral family Adenoviridae dsDNA
• Size 70-90nm
• Examples and diseases adenoviruses (see Fig. 1E)
  (respiratory infections, gastroenteritis,
  infectious pinkeye, rashes, meningoencephalitis)

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Pox Viruses

• double-stranded, circular DNA enveloped
  complex
• Viral family Poxviridae
• Size 200-350nm
• Examples and diseases smallpox virus (smallpox),
  vaccinia virus (cowpox), molluscipox virus
  (molluscum contagiosum-wartlike skin lesions

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Herpes and Hepadnaviridae

• double-stranded DNA enveloped polyhedral
  capsid
• Viral family Herpesviridae
• Size 150-200nm
• Examples and diseases herpes simplex 1 virus
  (HSV-1 most oral herpes see Fig. 1H), herpes
  simplex 2 virus (HSV-2 most genital herpes),
  herpes simplex 6 virus (HSV-6 roseola),
  varicella-zoster virus (VZV chickenpox and
  shingles), Epstein-Barr virus (EBV infectious
  mononucleosis and lymphomas), cytomegalovirus
  (CMV birth defects and infections of a variety
  of body systems in immunosuppressed individuals)
• Viral family Hepadnaviridae
• Size 42nm
• Examples and diseases hepatitis B virus (HBV
  hepatitis B and liver cancer)

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Picornaviruses

• ()single-stranded RNA naked polyhedral capsid
• Viral family picornaviridae
• Size 28-30nm
• Examples and diseases enteroviruses
  (poliomyelitis), rhinoviruses (most frequent
  cause of the common cold), Norwalk virus
  (gastroenteritis), echoviruses (meningitis),
  hepatitis A virus (HAV hepatitis A)

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Togaviruses

• ()single-stranded RNA enveloped usually a
  polyhedral capsid
• Viral family Togaviridae
• Size 60-70nm
• Examples and diseases arboviruses (eastern
  equine encephalitis, western equine
  encephalitis), rubella virus (German measles)

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- Strand viruses

• (-) strand multiple strands of RNA enveloped
• Viral family Orthomyxoviridae
• Size 80-200nm
• Examples and diseases influenza viruses A, B,
  and C (influenza)
• Viral family Bunyaviridae
• Size 90-120nm
• Examples and diseases California encephalitis
  virus (encephalitis) hantaviruses (Hantavirus
  pulmonary syndrome, Korean hemorrhagic fever see
  Fig. 1G)
• Viral family Arenaviridae
• Size 50-300nm
• Examples and diseases arenaviruses (lymphocytic
  choriomeningitis, hemorrhagic fevers)

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HIV

• produce DNA from () single-stranded RNA using
  reverse transcriptase enveloped bullet-shaped
  or polyhedral capsid
• Viral family Retroviridae
• Size 100-120nm
• Examples and diseases HIV-1 and HIV-2 (HIV
  infection/AIDS see Fig. 3A) HTLV-1 and HTLV-2
  (T-cell leukemia)

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Key steps in viral infections

• Adsorption
• Fusion/Uncoating
• Hostile Takeover
• Early Genes for Replication of Virus
• Late Genes for Proteins and Capsid
• Lysis

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Animations of adsorption

• Flash animation showing adsorption of an
  enveloped virus.
• Flash animation showing adsorption of a naked
  virus.
• QuickTime movie showing adsorption of an
  enveloped virus.
• QuickTime movie showing adsorption of a naked
  virus.

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Fusion and uncoating

• Flash animation showing penetration and uncoating
  of an enveloped virus entering by fusion.
• Flash animation showing penetration and uncoating
  of an enveloped virus entering by endocytosis.
• QuickTime movie showing penetration and uncoating
  of an enveloped virus entering by fusion.
• QuickTime movie showing penetration and uncoating
  of an enveloped virus entering by endocytosis

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Animations of Replication

• Flash animation showing replication of an
  enveloped virus.
• Flash animation showing replicationof a naked
  virus.
• QuickTime movie showing replication of an
  enveloped virus.
• QuickTime movie showing replicationof a naked
  virus. 

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Release of Virus

• Flash animation showing release of a naked virus
  by cell disintegration.
• QuickTime movie showing release of a naked virus
  by cell

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RNA Virus
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Global Pandemic
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Influenza

• Orthomyxovirus medium sized enveloped(-) sense
  RNA leads to recombination
• They have a segmented genome
• Broad host range
• Whales, swine,birds, and humans

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Antigens

• Influenza neuraminidase exists as a
  mushroom-shape projection on the surface of the
  influenza virus.
• It has a head consisting of four co-planar and
  roughly spherical subunits, and a hydrophobic
  region that is embedded within the interior of
  the virus' membrane.
• It is comprised of a single polypeptide chain

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Hemagglutinin

• HA binds to an as yet unidentified glycoprotein
  which is present on the surface of its target
  cells.
• This causes the viral particles to stick to the
  cell's surface. The cell membrane then engulfs
  the virus and the portion of the membrane that
  encloses
• It pinches off to form a new membrane-bound
  compartment within the cell called an endosome,
  which contains the engulfed virus.

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Avian flu

• Avian influenza is an infection caused by avian
  (bird) influenza (flu) viruses.
• These influenza viruses occur naturally among
  birds. Wild birds worldwide carry the viruses in
  their intestines, but usually do not get sick
  from them.
• However, avian influenza is very contagious among
  birds and can make some domesticated birds,
  including chickens, ducks, and turkeys, very sick
  and kill them.

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Infection

• Infected birds shed influenza virus in their
  saliva, nasal secretions, and feces.
• Susceptible birds become infected when they have
  contact with contaminated secretions or
  excretions or with surfaces that are contaminated
  with secretions or excretions from infected
  birds.
• Domesticated birds may become infected with avian
  influenza virus through direct contact with
  infected waterfowl or other infected poultry, or
  through contact with surfaces (such as dirt or
  cages) or materials (such as water or feed) that
  have been contaminated with the virus.
• Infection with avian influenza viruses

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Strains of Avian Flu
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Hepatitis B Virus

• Hepatitis B is a DNA Virus of the Hepadnaviridae
  family of viruses.
• It replicates within infected liver cells
  (hepatocytes ). The infectious ("Dane") particle
  consists of an inner core plus an outer surface
  coat.

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Hepatitis B antigens

• When the virus enters the body of a new host it's
  initial response, if it's gets past the immune
  system, is to infect a liver cell. To do this the
  virus attaches to a liver cells membrane and the
  core particle enters the liver cell. The core
  particle then releases it's contents of DNA and
  DNA polymerase into the liver cell nucleus.
• From within the cell nucleus the hepatitis B DNA
  causes the liver cell to produce, via messenger
  RNA surface (HBs) proteins, the core (HBc)
  protein, DNA polymerase, the HBe protein, HBx
  protein and possibly other as yet undetected
  proteins and enzymes.

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HbS Ag

• Hepatitis B Surface protein(s). (HBsAg) The
  outer surface coat composed of hepatitis B
  surface proteins is produced in larger quantities
  than required for the virus to reproduce. The
  excess surface proteins clump together into
  spherical particles of between 17-25nm in
  diameter but also form rods of variable length.
  In some cases these particles encapsulate a core
  particle and produce a complete, and infectious,
  virus particle that enters the blood stream and
  can infect other liver cells. The excess spheres,
  rods and also complete viral particles enter the
  blood stream in large numbers and are easily
  detectable. It does however take a while for
  these proteins to appear.

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Viral Structure
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Genes and Gene Products
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Viral Replication
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HBV
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Picornaviruses
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Polio virus

• Picornaviruses are non-enveloped.
• As a consequence, they are resistant to lipid
  solvents like ether and chloroform which would
  destroy an enveloped virus.
• Picornaviruses have an icosahedral capsid.
• .

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Poliovirus

• An enterovirus
• Enters the body through inhalation of water
• Used to be from swimming pools
• Causes encephalitis
• CNS involvement from mild to severe.
• Presents with fever, malaise,cnills, and
  neruological effects

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Rhinoviruses

• Picornaviruses replicate in the cell cytoplasm.
• Picornaviruses can replicate in an enucleated
  cell (a cell that has had its nucleus removed)
  because they only need host cell machinery and
  components that are present in the cytoplasm.
  They dont require anything found in the host
  cell nucleus

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Echoviruses

• Picornaviruses have a positive sense RNA genome.
• The picornavirus genome has the same sense
  (polarity) as mRNA.  The genome alone can use the
  host cell's machinery to make whatever is needed
  to replicate the virus.  If only genomic RNA is
  injected into a host cell, it is infectious and
  the cell produces progeny virions. 

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Rhabdovirus - Rabies
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Rabies

• Ss RNA virus
• Secreted in the saliva of infected animals
• Transferred by bite or scratch
• 75,000 cases if rabies occur around theworld in
  humans
• Virus travels on axons of motor or sensory
  neurons
• Spreads to brain with serious consequences

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National Rabies Management Program

• The development of a natural vaccine
• Research completed at Wistar Institute in
  Philadelphia, Pa.
• Vaccine is an oral type placed in natural
  habitats
• Food or bait is desirable to many animals such as
  fox, bear, otter, beaver, and racooons

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Distribution of Rabies in the United States