L1 Flashcards
Dimitri Ivanofsky showed that Tobacco Mosaic Virus (TMV) was able to
pass through a filter while bacteria could not (1892)
First electron micrograph of TMV in 1939
Martinus Beijerinck showed that the titer of TMV
increased after infecting a plant, proving TMV was not a toxin (1898)
Bacteria viruses (bacteriophage) discovered by
Frederick W. Twort (1915) while trying to grow vaccinia virus
Bacteriophage were instrumental in developing the field of
virology and expanding the field of biology
Foot and mouth disease
first animal virus) discovered (1898)
Yellow fever virus
(first human virus) discovered in 1901
Viruses are
obligate intracellular parasites
Viruses are not
autopoietic
Cellular origin
Proposes that viruses were once cellular components but over time they evolved separately.
Autopoietic origin
Proposes that viruses, once autopoietic entities, became dependent on cells for replication.
Attributes for Virus Classification
Virus particle structure Genome Replication features Serology Stability
Nucleocapsid
RNA or DNA in a core that is protected by a protein coat (capsid)
Virus is defined by the nucleocapsid structure
Nucleocapsid structural symmetry
helical
pelomorphic
Icosahedral
Nucleocapsid is comprised of repeating
protein subunits called capsomeres
Envelopes:
virus-modified cellular membranes acquired upon exit from host
Exposure to lipid solvents in the laboratory (e.g., alcohol, ether, acetone, Freon, etc.) renders enveloped viruses
noninfectious
Enveloped viruses may have nucleocapsids with
different structures
Smallest virus
18 nm
Largest
300 nm
Virus Genome DNA
double or single stranded
Virus genome RNA
RNA Double stranded Single stranded Plus sense (+)ssRNA Minus sense (-)ssRNA (polarity) Ambisense
Ambisense
Among the negative RNA viruses, ambisense RNA viruses or ‘ambisense viruses’ occupy a distinct niche. Ambisense viruses contain at least one ambisense RNA segment, i.e. an RNA that is in part of positive and in part of negative polarity.
Virus Genome Structure
Linear Circular Segmented Diploid Gene arrangement can change this
Virus Replication steps
Attachment Entry Transcription Translation Replication Assembly Release
Viral attachment is the
Binding of a virus receptor to a cellular receptor
Cellular receptors
Signaling molecules – induced cellular response to binding
Cell adhesion
Transport
Viral receptors usually do not
mimic cell receptor’s normal ligands
viral receptors typically are
Typically are spike like projections on particle surface
Viral creceptor smay require
a co-receptor- e.g., HIV (CD4, CXCR4)
Genetic engineering - can change receptor recognition
Integrin RGD sequence – used by Ad, engineered into lambdaphage
Pseudotyping particles – improve retroviral entry, VSV, Ebola, LCMV
Attachment is a major determinant of
virus tropism (host range)
Viruses infect essentially all known
forms of life
Specific host ranges, some can infect humans and animals (zoonosis)
Not shared across more divergent hosts (plants, bacteriae
Virus Replication – Entry - Pathways
Receptor rrrr3 mediated endocytosis
Direct penetration of plasma membrane
Enveloped virus
- viral entry
Membrane fusion
Best understood for influenza
HA protein – attachment & fusion
Receptor conformational change
Receptor conformational change
Low pH
Receptor induced
3Uncoating
involves nuclear replication nd cytoplasmic replication after uncoating
Nuclear replication
Genome and remaining protein coat (nucleocapsid) transported to the nuclear membrane
Delivery of genome to nucleus
Cytoplasmic replication
Release of the genome in cytoplasm
Transportation of the genome to intracellular site of replication
Many RNA viruses replicate in membrane associated complexes
dsRNA viruses never release their
genomic material from the entering particle
DNA viruses usually rely upon cellular
RNA polymerases
The genome of (+)ssRNA viruses can
serve as mRNA
Production of new transcripts can occur later using a (-)ssRNA template
(-)ssRNA, dsRNA viruses must bring their own
polymerase into the cell
All viruses need the cell’s
ribosomes to produce protein – no exceptions
Viral protein production can be regulated at the
transcript (mRNA) level or translation level
Structural proteins are produced in
high quantities
Non-structural proteins are only seen inside the
infected cell
(+)ssRNA Genome serves as template for
translation
(+)ssRNA
-Polymerase makes (-)ssRNA copy as
template for new genomes
(-)ssRNA
Virus particle must include the
viral polymerase
-ssrna Polymerase makes
messenger RNA for translation
Viral Genome replicates through full-length
(+)ssRNA intermediate
dsRNA
Virus particle includes viral polymerase
dsRNA induces innate
immune response so genome stays inside particle
mRNA synthesized in particle and exported to
cytoplasm
mRNA serves as
(+) strand in virus genome, (-) strand synthesized during assembly
ssDNA and dsDNA must gain access to
nucleus
Poxviruses are an exception, virion contains the necessary
RNA polymerase and the genome encodes the DNA polymerase for replication
ssDNA and dsDNA Prepare the cell for
DNA replication
Growth phase, dNTP production, replication machinery
Ensure genome ends are copied
ssDNA and dsDNA Prepare the cell for
DNA replication
Growth phase, dNTP production, replication machinery
Ensure genome ends are copied
Virus assembly
Package new genomes into functional particles
Localize structural proteins to aid assembly
Cellular viral “factories”
Virus Genome contains
packaging signals
Adenovirus –
empty protein coat imports genome
Reovirus –
RNA packaged during capsid assembly
Retrovirus –
preassembly on a membrane
Lysis
Best known for bacteriophage
Viral molecules that rupture cellular membrane
Weak Lysis
Depends on membrane breakdown after cell death
Budding (enveloped only)
Enveloped viruses use cell membrane as the outer coat of the virus particle
One step growth curve
Infect every cell at same time (MOI > 5)
Every cell dies at end of infection
Phases
Eclipse
Exponential growth
Plateau
Eclipse:
attachment and uptake
Exponential growth:
replication and assembly
Plateau:
cell death
Virus Replication - Kinetics Useful to assess:
Mutations
Cell entry
Process design
Time for one step growth
Start of infection to beginning of plateau
Bacteriophage – 30 min
Vesicular stomatitis virus (VSV): 6 hours
Vaccinia: 24 hours
Productivity
Measure amplification
VSV – 1:1000
Vaccinia – 1:100
Initial discovery
Disease in a host
Contaminant in cell culture
Confirmation
Purification of virus
Confirmation of disease
Animals, eggs or cell culture
Cell culture is preferred
Principle of Detection and Quantification Methods
Infectivity
Physical
Genome
Serological
Cytopathic Effect (CPE) infection assay
Cell Rounding
Syncytia Formation
Inclusion bodies
Fluorescent Focus Assay
infection assay
Infect cells
Expose virus antigen
Stain with labeled antibody
Count areas that fluoresce
Plaque Assay
infection assay
Cell monolayer
Inoculate with dilute virus
Infected cells die leaving a clear area – plaque
Infectious Dose
infection assay
ID50, IU50, TCID50
Systems: Tissue Culture, Eggs, Animals
Inoculate with different dilutions of virus
Calculate concentration based on number infected
Particle Assays
Electron microscopy
Hemagglutinin assay
Electron microscopy
Direct image of virus particles
Calibrate with latex bead standard
Hemagglutinin assay
Viruses that bind red blood cells (RBCs)
Mix constant number of RBCs with various virus dilutions
If virus concentration is sufficient, a matrix of RBCs and virus is formed
Matrix does not allow RBCs to pellet
Polymerase Chain Reaction
genome assay
DNA primer specific to virus
Amplify the gene
Very sensitive
Southern (DNA) & Northern (RNA) Blots
genome assay
Isolate DNA or RNA
Separate by electrophoresis
Use labeled DNA probe to detect
Serological Assays
Virus neutralization
Enzyme Link Immunosorbant Assay (ELISA)
Western (Protein) Blot
Virus neutralization
Antibody binding to virus can block infection
Virus concentration determined by amount of antibody needed
Enzyme Link Immunosorbant Assay (ELISA)
Antibody recognition of virus
Amplification by enzyme linked to antibody
Western (Protein) Blot
Separate proteins by electrophoresis
Probe proteins using an antibody
Viruses are obligate
intracellular parasites; use host cell’s replication processes to duplicate themselves
Every virus has a
nucleocapsid consisting of genetic material (RNA or DNA) and protein; some viruses are enveloped (coated with a host cell membrane)
Human viruses have diameters of
30-300 nm
Viruses are classified by the
genome, virus particle structure, and replication strategy
RNA viruses replicate in
cytoplasm
DNA viruses replicate in
nucleus