Module 1 Flashcards
what is a virus?
subcellular, infectious agents, consisting of nucleic acid (DNA or RNA) in a protein coat
what is a virus?
obligate intracellular parasites
viruses’ one goal
to replicate themselves
viruses
type of infectious agent
virions
individual virus particles
virus delivery system
protein coat
virus payload
nucleic acid
why do we study viruses?
important pathogens, infect all forms of life, transfer genes between organisms to drive evolution, play a large role in ecological, useful for preventing and curing diseases, providing insight to basic mechanisms
virology is a young science
around 120 years old, hippocrates rationalized plagues caused by small organisms
viruses are filtrable agent
viruses smaller than bacteria passed through filter, weren’t grown on culture but would infect animals
viruses could …
diluted and still cause disease, can regain its strength through replicates, can be passed multiple times
viruses fail to propagate in solutions
further study hampered by lack of experimental system
Koch’s postulates
virus don’t grow on culture so they don’t follow koch’s postulates
poliovirus replication in cell cultures
ender, wellers, and robbins propagate poliovirus in human cell cultures in primary embryonic skin
study of viruses has an impact on molecular and cellular biology
gene expression, DNA replication, RNA splicing, cellular oncogenes
smallpox eradication
1958-1979, first and only human infectious disease to be eradicated
eradication of rinderpest
2nd Virus, 2011, eradicated in cattle
generic viral structure
nucleocapsid and envelope
nucleocapsid
all viruses have a capsid and nucleic acid
envelope
viral proteins embedded, some viruses have envelopes
viruses alive?
made of the same material as cells, replicate, evolve, some metabolize
viruses dead?
do not have cells, cannot reproduce independently, lack ribosomes, do not typically metabolize
viral genome
RNA or DNA contains the information needed to initiate and complete an infectious cycle within a susceptible or permissive host cell
where do viruses package their genomes?
inside a protein shell
all viruses can establish themselves in a host population, so as to ensure virus survival
true
an infectious cycle includes…
attachment, entry/uptake, production of viral mRNA and proteins, genome replication and assembly, release of new particles
how common are viruses?
most abundant for of “life,” most common thing that replicates
in seawater?
10-50 million phage/ml
viruses play a major role in …
carbon and oxygen cycles that regulate the atmosphere
how much oxygen is generated by marine microbes through photosynthesis
50%
how many marine microbes are destroyed a day by viruses?
20%
human body
10 trillion cells are products of 23,000 genes
microbiome
100 trillion bacteria, viruses, and fungi and 3 million non-human genes
all cells are infected with viruses
true
how much of your DNA is made up of old and new retrovirus genomes
8%
are retroviruses passed to human offspring
yes
everyone has herpes
each of you is infected with at least two types of 9 known herpesviruses, once infected you are infected for life
virus characteristics
small, DNA or RNA genome, small genome size (3000 nt - 1.2 million bp), genomes associated with protein, only replicate in living cells
virus particle
virion capsid alone or capsid and lipid bilayer envelope
how many rhinoviruses can fit on the head of a pin?
9.069 billion
mimivirus
kind of metabolize, could have been caused by reductive evolution
how many mimiviruses can fit on the head of a pin?
2.27 x 10^7
vertebrate viruses
RNA genomes outnumber DNA genomes 2 to 1
viral genomes
compact/economical, ~1 protein/1000 nt
mammalian genomes
~ 3 billion nt, ~ 1 protein/ 100,000 nt
gene products and regulatory signals for replication of the viral genome
encoded in viral genomes
gene products and regulatory signals for assembly and packaging of the viral genome (capsid formation)
encoded in viral genomes
gene products and regulatory signals for regulation and timing of the replication cycle
encoded in viral genomes
gene products and regulatory signals for modulation of host defenses
encoded in viral genomes
gene products and regulatory signals to spread to other cells and hosts
encoded in viral genomes
genes for complete protein synthesis machinery
not encoded in viral genomes
genes for proteins involved in membrane biosynthesis
not encoded in viral genomes
classical centromeres or telomeres
not encoded in viral genomes
enzyme systems that produce nucleotides, amino acids, carbohydrates and lipids
not encoded in viral genomes
metabolic enzyme systems that generate useable chemical energy
not encoded in viral genomes
50-90% of virion mass is made up of?
protein
capsid variation
limited number of particle design
structure of virion functions
protects nucleic acid from nucleases, environment, and shearing, contains elements to recognize target cells, built in system for genome release at correct time and location, includes enzymes essential for infectivity
virions are metastable
protection of the genome is stable, coming apart for infection is unstable
virus particles are morphologically diverse
true
filamentous viruses
helical symmetry, nucleic acid core with 1-2 subunit tube
isometric viruses
icosahedral symmetry, 20 faces and 3 axes of symmetry
complex viruses
maximalist approach to metastability, round shaped
what percentage of virus taxa have icosahedral capsids?
60%
icosahedron characteristics
largest ratio of volume to surface area, thermodynamically favorable, maximum enclosed volume for shells
why is icosahedral or helical symmetry beneficial?
genetic economy and efficiency and greater stability due to symmetrical arrangement
there are a limited number of ways to achieve icosahedral or helical symmetry
true
nonenveloped icosahedral
animals, plants, bacteria, and vertebrate viruses
nonenveloped helical
plants and bacteria viruses
enveloped icosahedral
animals, bacteria, and vertebrate viruses
enveloped helical
animals, plants, bacteria, and vertebrate viruses
head tail
bacteria virus
complex
animals and vertebrate viruses
do viruses have enzyme systems that produce nucleotides, amino acids, carbohydrates and lipids
no, acquired from host cells
do viruses have enzyme systems that generate useable chemical energy
no, acquired from host cells
do viruses have ribosomes, transfer RNA and enzymes needed for protein synthesis
no, viruses are completely dependent on host for protein synthesis machinery
do viruses have membranes to localize and concentrate cellular macromolecules (organic and inorganic ions)
no, acquire from host cell membranes
animal viruses classified by…
replicative strategy, structure, genome, and host
animal virus classification: structure
icosahedral, helical, or complex, enveloped or non-enveloped
animal virus classification: genome
DNA or RNA, single or double stranded, positive or negative sense
baltimore classification of viruses
basis is pathway from genome to early mRNA, viral genomes must make mRNA that can be read by host ribosomes
class I
double stranded DNA
class II
single stranded DNA transcribed to double stranded DNA
class III
double stranded RNA
class IV
positive sense single stranded RNA transcribed to negative sense single stranded RNA
class V
negative sense single stranded RNA
class VI
single stranded RNA-RT transcribed to DNA or RNA then into double stranded DNA
class VII
double stranded DNA-RT makes RNA copy and retrotranscribed into DNA
?s for baltimore classification
is the genome RNA or DNA
is the genome double or single stranded
if ssRNA, is it positive or negative sense
does the virus use reverse transcription
international committee on taxonomy of viruses classification factors
range of characteristics, scheme of order, family, subfamily and genus, concept of species is complex and debated
ICTV classification of characteristics
virion morphology and size, nucleic acid type, presence or absence of specific genes, host range, and phylogenetic groupings
order suffix
-ales
family suffix
-idae
subfamily suffix
-inae
genus suffix
-virus
type member example
measels virus
viral phylogeny
align viral sequences of related viruses to interpret relationship
viral phylogeny: root
presumed ancestor
viral phylogeny: scale
number of changes per length of sequence
viral phylogeny: branches
lineages
viral phylogeny: clade
branch that represents all viruses with a common ancestor
viral phylogeny: tips or leaves
individuals sequenced
viral phylogeny: measure of support
probability that sequences cluster together better than other sequences
viral phylogeny: nodes
ancestors may be inferred
serotype
a system of grouping viruses based on the type of surface antigens present
serotyping is generally determined by …
reactivity of viruses with antibodies in serum from individuals infected with specific virus isolates, but this is hard to set up
serotype antibody reactivity
measured by virus neutralization, ELIZA, and hemagglutination inhibition
serotyping and PCR
once system is established, PCR can be used due to known coding sequences
origin of viruses: virus early hypothesis
start small and acquire genes to gain complexity
origin of viruses: regression hypothesis
start large and loose genes by relying on other cells for resources
origin of viruses: escaped genes hypothesis
host genes escape and replicate
accepted origin of viruses
diversification of replicators and replication strategies from ancestral RRM, acquire protocapsid genes by selfish replicators from primitive cells, evolution of modern cells
RRM
RNA-recognition motif domain
did viruses start out as RNA?
RNA originally then transition to DNA based world
no single progenitor for origin of viruses
some retroviruses are 450 million years old, some may have originated billions of years ago before cells
once viruses were formed
subject to evolutionary pressures through mutation, recombination, and reassortment. the host provides powerful selective pressure
what is the viruses main goal?
replication
do viruses evolve for virulence?
no, they evolve for replication
anything that occurs during virus infection is…
supportive of virus replication, a side effect of virus replication, or a result of host response to virus infection
viruses do not _____
divide
burst size
the number of virions produced from infection of a single cell
cellular stages of virus replication cycle
enter cell and translocate to the site of replication, replicate genome and produce mRNA, generate viral proteins, assemble pyrogeny virus, emerge from cell
extracellular stages of virus replication cycle
evade host defenses, disperse and persist in the environment
virus replication cycle pathway
attachment, entry, uncoating, viral genome expression, replication of viral genome, assembly, maturation, release
virus fitness
a complex property, the replicative ability of a virus to its environment
fitness in vitro
determined by comparison of growth rates and viral yields
fitness in vivo
difficult to measure under natural conditions, infection of complex organisms is large interacting populations
relative fitness
ratio of fitness between two viral variants
relative fitness =1
fitness is neutral
relative fitness <1
reduced fitness
relative fitness >1
increased fitness
what kind of population is required for replicative fitness?
a stead state equilibrium population with a large number of virions/genotypes
virus evolution is driven by
genetic variation and environmental selection
genetic variation occurs through
mutation, recombination, and reassortment
environment selection occurs in
the cell, the host, and the environment
the _____________ of a viral genotype in a given environment determines viral fitness
replicative success
genetic variation comes from
errors in replication of the viral genome
high mutation rate
increases the amount of genetic variability available for selection
mutation
an alteration in the nucleic acid sequence of the viral genome
large population size
increase the probability of the occurrence of an advantageous genetic change
short generation time
lessens the time required for advantageous genetics to become selected
viruses have high mutation rates
true
mutation rate
genetic changes in individual viruses
substitution rate
genetic changes within a population (selection)
PRRSV virus
mutation rate not particularly high, disparity between mutation and substitution rate is likely due to selection
effects of single base mutations
no effect, (most as far as we know)
single base mutations: alter animo acid sequence
non-synonymous, codon changes, alternate start/stop, splicing
nonsynonymous vs synonymous
nonsynonymous does not always have greater effects
single base mutations: affect transcript abundance
promoter efficiency and message stability
single base mutations: influence protein translocation/modification
codon usage bias, signal sequence, glycosylation, ubiquitination
few mutations, nonsynonymous and synonymous, actually increase relative fitness
true
nonsynonymous mutations are more _____ than synonymous mutations
lethal
mutations in viral polymerases that reduce the frequency of errors
do not have a selective advantage over wild type, lower mutation rates are neither advantageous nor selected in nature, mutants are often less pathogenic
high mutations are ____ during virus evolution
selected for
mutation is _______ for viral population (within limits)
good
viral recombination and reassortment
requires coinfection of a single cell with two different viruses
viral recombination requires
coinfection of a single cell with 2 different viruses and sufficient sequence homology
recombination are generally random but…
hot spots may exist and fitness selects successful recombinants
genetic drift
polymerase errors lead to nucleotide substitution during genome replication, small changes occurring over time, viral coded polymerase generates a lot of copy errors, may be small changes, but can have big impacts due to large number and short life cycle
why do NA virus polymerases generate a lot of copy errors?
RNA viruses totally lack proof reading activity and have lower fidelity
genetic shift
large scale changes due to recombination or reassortment of gene segments between two related viruses, big changes over short periods, requires coinfection of a cell with two dissimilar viruses
genesis of the 2009 swine influenza A virus (H1N1)
multiple reassortment events, involves three host species, four parental viruses, change antigenic phenoytpe, change virulence
effect of genetic drift, bottlenecks and founder effects on genetic diversity
the smaller a population is, the greater the effect of genetic drift on genetic diversity
viral quasispecies are the _____ in RNA viruses
norm
quasispecies concept
virus populations exist as a dynamic distribution of nonidentical, but related replicons called quasispecies
the ability to produce a quasispecies may also virus populations to __________ encountered during spread between hosts, within organs and tissues, and ___________
respond to the different environments, and respond to the pressure of the host immune response
quasispecies provide …
an interpretation for the extensive plasticity, both genetic and phenotypic, displayed by many viruses
error prone replication leads to formation of …
dynamic mutant distributions
viral quasispecies develop due to _________ and are shaped by _________
errors in replication, selection
non tree like patterns are the result of recombination, represented by
a closed lip
quasispecies: viral infection in a host consists of initiated by a _________ not a single virus genome
population of genomes
quasispecies: the large number of pyrogeny are the product of _______________
selective forces inside the host acting on the population
quasispecies: the survivors that can infect a new host reflect _________________
selective forces outside the host acting on the population
quasispecies wild type
a bunch of different genomes
quasispecies act as ________ of mutants for selection to act upon
a phenotypic pool
quasispecies have _____ and have a record of past genome dominances, ready to readapt to previously experienced selective pressures
memory
quasispecies memory is reduced by …
bottlenecks
quasispecies members ______ within the swarm
interact
how do quasispecies members interact?
cooperation, complementation, and interference
survival of the fittest
a rare genome with a particular mutation may survive a selection event, and this mutation will be found in all pyrogeny genomes
survival of the survivors
linked but unselected, mutations get a free ride
the product of selection after replication is a …
new diverse population that shares the selected and linked mutations
hepatitis C virus passage in a constant environment
mutations accumulate in the population over time
dominant
high frequency in the virus population
frequency of individual mutations in the virus population
some persist, others come and go, dynamic
error threshold
mutation is advantageous but selection and survival balances genetic fidelity and mutation rate
error threshold
the tipping point between mutation rate and survival
exceeding the error threshold results in
loss of infectivity, “death”
Too far below the error threshold results in
viruses inability to produce enough mutations to survive selection
RNA viruses evolve ________ the error threshold
close to
DNA viruses evolve _______ the error threshold
far below
viruses must achieve a ________
balance between mutation and error threshold
antiviral ribavirin and poliovirus
ribavirin is a guanosine nucleoside analog used to stop viral RNA synthesis and viral mRNA capping. Pairs with uracil or cytosine, including mutations in RNA dependent replication
recombination ______ error catastrophe
counters
poliovirus RdRp L420A
recombination defective, exacerbates ribavirin induced error catastrophe, recombination required to counter error catastrophe, reassortment may also accomplish in other viruses
reduced bottlenecks
causes the accumulation of mutations exceeding error threshold leads to decreased replicative fitness
mutations that improve replication lead to
increased replicative fitness
repeated bottlenecks lead to
accumulation of deleterious mutations leading to decreased replicative fitness
muller’s ratchet
small asexual populations decrease in fitness over time if the mutation rate is high, mutations that exceed the error threshold accumulate during replication and fitness decreases
why is fitness reduces?
muller’s ratchet, replicating RNA viruses producing many mutations close to the threshold, restricting population growth to serial single founders under otherwise nonselective conditions, so many mutations accumulate exceeding the threshold that decrease fitness
sequence space
refers to every possible combination of a give sequence
sequence space is theoretically a
vast multidimensional hypercube connecting all possible combinations
sequence space for 400 bases with 4 alleles
4^400 = 7 x 10^240 different genotypes
fitness landscapes
systemic fitness and sequence space
fitness landscapes: peak
higher rate of survival, will be present in the quasispecies, evolve
most changes in genome are …
not beneficial
fitness
the likelihood of a gene, organism, or other unit of study to make it into the next generation
virus and prey relationships
not antagonistic, one leads and one follows
neutral sequence space
extent of sequence space in which a sequence change may occur without a fitness cost
what defines neutral sequence space?
selective pressure from host factors
red queen hypothesis
infectious agents and their hosts coevolve as one changes the other adapts to that change
virus remain _____________ to their host as the host themselves evolve
associated and highly adapted
viruses infecting new species are …
initially poorly adapted
what do cells provide for virus replication?
building blocks, metabolism to generate energy, protein making machinery, membranes
cell building blocks for virus replication
nucleotides, amino acids, carbohydrates, and lipids
cell metabolism to generate energy for virus replication
ATP
cell protein making machinery for virus replication
ribosomes, transfer RNAs, enzymes
cell membranes for virus replication
localize replication, concentrate building blocks, supply the lipid bilayer needed for envelopes
propagation of viruses in animals
originally used laboratory animals and embryonate eggs
explants
bits of tissue maintained in culture
growth of viruses in embryonated eggs
used for flu vaccines
enders wellers and robbins discovered
ability of poliomyelitis viruses to grow in cultures of various types of tissue
susceptible cell
functional receptor for a given virus, the cell may or may not be able to support viral replication
resistant cell
no receptor for a given virus, it may or may not be able to support viral replication
permissive cell
the capacity to replicate a given virus, it may or may not be susceptible, no receptor
what is the only cell that can take up a virus particle and support its replication?
a susceptible and permissive cell
primary cells
heterogenous (many cells present), closest to animal cells, technical hassle
diploid cell strain cells
relatively homogenous, further from animal cells, technically less hassle
continuous cell line cells
immortal, most homogenous, genetically weird, furthest from animal cells, pretty easy to grow cells
primary cell culture technique
collagenous treatment of mince tissue, filtered and pellet, harvest supernatant and grow on dish
consequences of primary cell passage
lose cell population heterogeneity, cell population dies out
all continuous cell lines are not ______
alike
virus induced cytopathic effect (CPE)
visible morphological changes in cell cultures caused by viral infections
CPE
death of cells, rounding up and detachment, swelling and clumping, vacuolization, syncytia, inclusion bodies
syncytia formation
infected cell fusion into a multinucleated cell
viral inclusions
aggregates of viral protein in the cell that stain differently
types of viral inclusions
intranuclear and cytoplasmic
hemadsorption
for non-cytopathic viruses that produce hemagglutinins (viral proteins that adhere to RBCs)
viruses that use hemadsorption
influenza, mumps, and parainfluenza
focus forming assay for hepatitis C virus
uses IHC or IFA instead of CPE to determine endpoint of titration
infective assays
plaque assay, TCID/CCID, hemagglutination, hemadsorption
microscopy methods
electron microscopy, immunohistochemistry, in situ hybridization
nucleic acid methods
PCR and sequencing
serologic antibody based methods
ELISA and lateral flow
detection and quantification of viruses
1952, Dulbecco and Vogt developed a plaque assay for animal viruses which transformed animal virology from a descriptive to a quantitative science
virus titration - plaque assay
mix virus dilution with cells and overlay with agarose to limit virus diffusion, remove agarose stain remaining cells with crystal virus to visualize plaques, virus titer determined by counting plaques and multiplying by the dilution factor, each plaque is the result of infection by one virion
tissue culture dose
concentration of virus it takes to produce cytopathic effect in 50% of the wells infected with virus
virus quantification by TEM
add latex beads and virus estimated through electron microscopy
TEM advantages
can identify type of virus by morphology, does not depend on virus growth, detect viruses in tissue or dirty samples such as feces and urine
TEM limitations
relatively low sensitivity, sample is dead, cannot differentiate between viable and nonviable virions, expensive equipment
TEM detection to viruses in dirty samples
can analyze directly from vesicular fluid, feces, and urine
TEM identification of virus by morphology
size, capsid symmetry, envelope, and other features
particule to PFU ratio
count virus particles by EM, determine PFU by plaque assay, divide number of particles by number of PFUs, variable is always >1, not all virions are infections
immunohistochemistry and immunofluorescence
antibody binds to viral antigens, enzyme (IHC) or fluorescent (IFA) label on antibody, cell culture or tissue samples
in situ hybridization
detects viral nucleic acid, may be direct or indirect
PCR
geometric amplification of DNA segments, very high sensitivity -1 copy of genome, need to know target sequence, cannot tell if virus is infection
sequencing for virus detection advantages
no assumptions, sequence info for culture required, can get strain phylogenetic and epidemiologic info directly, can identify previously unknown viruses, may detect multiple pathogens
sequencing for virus detection limitations
cost is relatively high, low sensitivity compared to PCR, high degree of skill required, no viability info
hemagglutination
used to estimate titer
what does ELISA stand for?
enzyme linked immunosorbent assay
ELISA
relatively fast, high specificity, generally high sensitivity, can be quantitative, lots of different test architectures, used to detect specific antibodies
what is important to know about ELISA?
how it was done changes the way you interpret it
common ELISA test architectures for virus detection
direct assay, indirect assay, capture assay sandwich
Lateral flow test for virus antigen detection
good sensitivity
virus one step growth curve
every cell in dish synchronously infected
multiplicity of infection
the ratio of infectious virus to susceptible and permissive cells
MOI
number of infectious units divided by the number of susceptible cells, infection depends on random collision of virion and cells, infection predicted by a poisson model
