6_Basic Virology I & II Flashcards
characteristics of viruses
- obligate intracellular parasits
- metabolically INERT
- NOT sensitive to Abx
- replicate by unique method
- DNA or RNA genome
viral size:
- how to determine
- typical size/largest known?,
- determined by electron microscopy
- ~10-400 nm, w/ largest being 1 micrometer (pandoravirus or megavirus)
largest viruses identified?
w/ largest being 1 micrometer
pandoravirus or megavirus
components of viruses
- naked vs. enveloped
- enveloped has an extra piece that “covers it up”
important terms for chemical composition and structure
(G CENN)
- Genome
- Capsid - capsomere
- Enveloped vs. naked - peplomers
- Nucleocapsid
- Non-structural proteins
Classification by viral genomes?
- DNA
- ds-DNA, vs. ss-DNA (single/double)
- Circular vs. linear
- RNA
- ds-RNA
- ss-RNA, vs. - ss-RNA
- Circular vs. linear
capsid:
- define,
- fxn,
- structure
- protein structure surrounding viral genome; *determines the shape of virus
- fxn: introduces viral genome into host cell
- symmetry; 2 basic shapes –> determines shape of virus
- Icosahedral (cubic)
- Helical
some examples of helical viruses,
and icosahedral?
helical - TOBACCO MOSAIC, RABIES
icosahedral - HERPES SIMPLEX, POLIOVIRUS
capsomere:
define and fxn
- subunit of the capsid; comprised of few protein subunits that are repeated
- self-assemble to form the capsid
nucleocapsid:
define
viral capsid w/ its enclosed nucleic acid (DNA/RNA)
enveloped vs. nonenveloped
key differences
-
many enveloped viruses are transmitted human-human in respiratory droplets (e.g. influenza, measles, rubella, varicella-zoster)
- enveloped are also transmitted by direct contact (blood, sexually)
- *enveloped are more sensitive to heat/drying/detergent/lipid solvens
which can survive in the environment longer?
(enveloped or nonenveloped)
What is this viruses’ transmission?
nonenveloped viruses can survive in environment longer and can be transmitted by fecal-oral route
viral envelope:
define
- derived from host during replication – composed of host cell lipids and viral glycoproteins
- *not present in all viruses
peplomer:
define, fxn,
where found?
- glycoprotein “Spike”, binds specific receptors on host cells
- host specificity
- viral infectivity
- on viral envelope OR naked capsid
non-structural proteins:
where found?
functions?
- packaged w/in the virion
- fxns: (ETER)
- establish infection
- transcribe viral genome
- escape host defense mechanism
- replication
in which systems can viruses be cultivated?
- animals
- embryonated eggs
- cells and organ cultures
poliovirus:
different models used to study
- HeLa cells: used to develop adn test Salk’s poliovirus
- PVR transgenic mouse: test nuerovirulence of poliovirus vaccine
- cells from African green monkeys: used by salk and sabin to develop vaccine (SV40 foun in injected vaccine)
Types of cultures for viruses
- Primary cells
- may have several cell types
- limited life span (5-20 cells divisions, 30-35 passages)
- maintain cell differentiation characteristics of original tissues
- Continuous cell lines
- usually derived from tumors
- primary cell culture have undergone mutations or transformed to become “immortal”
cell culture of virus:
function
method
- fxn: allows for controlled virus growth for study/virus manipulation; important in vaccine development
- method
- cells inoculated into flask/dish w/ medium
- cells divide to cover surface of flask/dish
- add virus to infect cells
reassortment:
define
mixing of genetic material of a species into new combinations in diff’t individuals
(pandemic flu strains were caused by reassortment b/w avian virus and human virus)
cell lines that can be used?
- Human cell lines: HeLa cells
- cervical cancer cell
- oldest (1851) and most commonly used immortal cell line (polio vaccine)
- Fetal cell lines
- obtained in 1960s
- used for vaccines - HepA, Varicella, Zoster, Rubella, Rabies
one-step growth experiment
how you quantitate life cycle of lytic viruses
due to “one-step growth curve”
how to quantify plaques?
- count plaque forming units to quantify amount of infectious virus you have
- 1 PFU = 1 infectious virion
- Process
- make serial dilutions to cell monolauer
- plaques develop in the cell monolayer
other important ways to detect virus
- hemagglutination assay
- cell culture
- cytopathic effect (CPE)
- immunofluorescence
- inclusion bodies
- Electron microscopy
- ELISA
- RIA (radioimmunoassay)
- PCR
hemagglutination:
define
- Hemagglutinin on the surface of influenza virus binds to the sialic acid receptors of red blood cells
- creates a lattice structure.
- The agglutinated lattice maintains the red blood cells in a suspended and shows as a reddish solution
immunofluorescence:
define
- technique using immunofluorescent tag
- look under certain filters
- surface antigen vs. nuclear antigens
cytopathogenic effects (CPE)
define, function
- Cellular damage caused by virus infection/replication observed in cell culture
- Can be diagnostic in biopsied/autopsied tissues
Cytomelagovirus inclusion bodies:
define
- CMV is a DNA virus, therefore has a nucleus
- So if cell is infected by CMV –> shows multinucleated giant cells,
- aka “Owl’s sign”
Rabies virus inclusion bodies:
define, name, staining
- (-)ssRNA virus, found in cytoplasm
- Negri bodies
- stain eosinophilic
syncytia:
define
Fusion of HIV infected macrophages and/or microglia is a common feature in HIV encephalitis (HIVE)
where does DNA viruses accumulate?
what about RNA viruses?
DNA virus –> in nucleus
RNA virus –> in cytoplasm
virus classification:
baltimore vs. other methods
Baltimore classification
- type of nucleic acid (genome)
- strandedness of genome
- polarity of RNA of genome
- method of replication
Other, using above PLUS…
- symmetry of capsid
- presence of envelope
- other criteria (i.e. # of capsomeres, size, disease, etc)
Baltimore classification:
why helpful?
- a helpful guide for mechanisms of virus replication, –> based on (+)mRNA production required of all viruses for replication
- (viruses in a given category will behave similarly)
how does viral replication differ?
Based on:
- Type of genetic material
- DNA - assemble in nucleus, EXCEPT poxvirus
- RNA - assemble in cytoplasm
- Polarity of RNA genome
- Presence/absence of envelop
- Requirement for reverse transcription
what must all viruses produce?
All Viruses must produce mRNA that can be translated by cellular ribosomes.
The unique pathways from various viral genomes to mRNA define specific virus classes on the basis of the nature and polarity of their genomes
General virus replication cycle:
high level
- Absorption
- Penetration
- Uncoating
- Replication of genome
- Maturation
- Release
General virus replication cycle:
absorption
•Highly specific – requires the interaction of a unique protein on the surface of the virus with a specific receptor site on the surface of the cell.
General virus replication cycle:
Penetration
•occurs by one or more processes
- Enveloped viruses fuse their envelope with the membrane of the host cell
- Local digestion of the viral and cellular membranes, fusion of the membranes, and concomitant release of the nucleocapsid into the cytoplasm
- Naked viruses bind to receptor sites on the cellular membrane, digest the membrane, and enter the cytoplasm
- Naked and enveloped viruses can be ingested by phagocytic cells
- Virus enters the cytoplasm enclosed in a cytoplasmic membrane derived from the phagocytic cell
General virus replication cycle:
Uncoating
Replication of Genome
-
Uncoating
- Cellular proteolytic enzymes digest the capsid (occurs in cytoplasm)
-
Replication of genome
- Processes are complex and variable and depends on the nucleic acid type
What is the difference between an abortive virus and a defective virus?
Abortive infection: Virus enters the host-cell but cannot successfully complete replication (non-productive infection).
- Cell is non-permissive
- Virus may be defective
- Actions of IFNs
Defective virus: Deficiency in some aspects of replication. Lack certain genes for replication and requires the help of another virus.
Describe the replication of a DNA virus,
and where it is found?
- Attachment and entry
- Translation and Genome replication
- Assembly and relase
“DNA-containing envloped virus, leaves the envelope, inserts into nucleus, replication occurs in nucleus, and leaves cell”
Host DNA transcription and translation:
key concept about where processes occur
- Messenger RNA (DNA-dependent RNA-polymerase is needed to do the work in the nucleus)
- In the cytoplasm, you need a ribosome for translation to occur
Double-stranded DNA viruses:
mechanisms for synthesizing proteins
*NEEDS THE HOST TO REPLICATE
•DNA –> DNA : Replicate in the host nucleus and follow the same patterns as those that already exist in cells
- highly dependent on a cellular state permissive to DNA replication (cell cycle)
- may induce the cell to forcefully undergo cell division, which may lead to transformation of the cell and, ultimately, cancer
- Ex., adenoviruses
•DNA –> RNA –> protein and replicating DNA
- Less common (Ex., Hepatitis B)
what other enzymes may be needed for ds-DNA virus replication?
need cellular polymerases, usually;
some viruses encode their own replication factors
(e.g. adenoviruses, herpes virus)
Where do Poxviruses replicate w/regards to cell?
Implication of this?
replicate in cytoplasm –> therefore carry their own polymerase w/in the virus particles
Are there exceptions to the rule:
DNA virus –> nucleus
RNA virus –> cytoplasm
YES!
DNA virus: Poxvirus replicates in cytoplasm
RNA virus: Orthomyxovirus and retrovirus (HIV) replicates in nucleus
Do all RNA viruses encode a RNA-dependent RNA polymerase?
All of them, EXCEPT RETROVIRUSES,
Retroviruses encode an RNA-dependent *DNA polymerase, instead
difference between positive and negative polarity of ssRNA
positive polarity: RNA w/ essentially same base sequence as mRNA
vs.
negative polarity: RNA sequence that is complemetary to the mRNA
what is required for replication of:
(+) ss RNA viruses
- can use RNA genome directly as mRNA
- Requires: RNA-dependent RNA polymerase to copy the viral genome
what is required for replication of:
(+) ss RNA viruses
- viral mRNA is synthesized by RNA-dependent RNA polymerase by using negative strand as template
- viral RNA polymerase is carried w/in viral particles
replication of:
ds RNA viruses
- **(+) RNA strand CANNOT be translated as part of RNA duplex
- viral mRNA is synthesized by a RNA-dependent RNA polymerase using the (-) strand as template
- viral RNA polymerase is carried w/in the viral particles
segmented RNA gemones:
define, and example
- there are several fragments of RNA that make a complete viral genome
- Ex: influenza virus
- 8 segments of viral RNA –> each segment can produce at least 1 protein
- these segments carry all the info needed to make new influenza virus particles
Retrovirus:
classification,
how does it replicate?
- enveloped, ssRNA, (+)
- requires reverse transcription before replicating –> integrates into host DNA (provirus)
virus-cell interactions
- abortive infxn
- productive infxn
- latency
- transformation
host response to virus infection
- cell-mediated immunity
- antibody
- interferon
early immune responses to infxn
what is the first line of defense against viral infection?
INTERFERONS (IFNs)
How does IFN affect viral replication?
IFN-a and -b
- Induced in response to accumulation of dsRNA in viral infections
Interferes with virus production
- Degrades mRNA
- Inhibits protein synthesis
How do interferons affect neighboring cells?
- Acts on non-infected cells to induce the “antiviral state”
- results in changes in production of a large number of proteins
which is first?
antibodies or cell-mediated immunity?
define each
Cell mediated immunty –> then antibodies
types of vaccines?
- live/attenuated virus
- viral subunit
- killed/inactivated virs
- DNA
- what types of immunity are induced by each?
- which are safest for immunocompromised patients?
live attenuated vs. killed/inactivated?
Live/attenuated is preferable bc it raises immune response to all protective antigens
compare live vs. killed vaccine for the following categories
viral subunit:
how is it prepared?
fxn?
how do DNA vaccines work?
Gene gun:
purpose,
how it works?
- DNA vaccine delivery
- Ballistic acceleration pDNA-coated gold (or tungsten) micro-fragments into target cells
- Compressed gas (He)
- Force allows cell penetration
- Saline injections require 10µg – 1mg pDNA to elicit an effective immune response
- Gene gun requires ~ 0.2 – 20µg
Advantages of DNA vaccines
which type of vaccine has vector w/ “no protein components to elicit an immune response”?
DNA vaccines
what are the possible problems associated w/ DNA vaccines?
what can DNA vaccination induce the production of?
•DNA vaccination may induce IgG anti-DNA autoantibodies
- Magnitude and duration of this response appears to be insufficient to cause disease in animal models
- May be specific to vector used
- Preclinical studies suggest systemic autoimmunity is unlikely
uses of viruses in gene therapy
- can use viruses as a delivery system
- either ex-vivo or in vivo gene editing/therapy
