Exam One Flashcards
Virion
complete extracellular infective form of a virus
Virus
includes the virion of inactivated virus particle, or viral nucleic acid and protein in the infected cell
Viriod
infectious particle smaller than viruses, agent of some plant dzs
Where will viral attachment proteins be found
envelope or capsid (if the virus is naked)
Nucleocapsid
nucleic acid + capsid
Incomplete virion
empty capsid
Defective virus
Can’t replicate due to mutations or errors, needs helper virus
Episome
extra-chromosomal genetic element
Provirus
viral DNA is integrated into host cell chromosome (can be transmitted to daughter cells)
Cubic Symmetry/Icosahedral
12 corners/verticies, 20 facets (triangula faces and 30 edges.
Helical structure
capsomeres + nucleic acids self-assemble, impossible for incomplete virions to form (all helical viruses are enveloped)
Complex structure
Made up of several parts w/ seperate shapes and symmetries. Think Pox Virus
Capsid functions
structural symmetry nucleic acid production attachment of naked viruses contains enzymes (reverse transcriptase) Detemines antigenic characteristics
How is the envelope/lipid bilayer acquired?
budding
What do Glycoprotein peplomeres/spikes do?
receptor binding
What do fusion proteins do?
Viral entry/release
What do matrix proteins do?
recognition site and add rigidity
How can the lipid bilayer be disrupted?
Drying, acid, heat
Solvents (ether or chloroform)
Detergents (sodium deoxcholate)
T/F all viral genomes are haploid
true, only contain 1 copy of each gene
Monopartitie
all genes in 1 choromosome (all DNA viruses)
Multipartitie/segmented
genes distributed among several chromosomes and can undergo genetic reassortment
Which sense RNA strand is infectious if injected into host cell? (+/-)
+
- sense RNA not infectious
Which is more stable, naked or enveloped viruses?
Naked
Viral proteins are denatured at what temp?
55-60
T/F acid leads to reversable dissassembly of the capsid
F, can also be irreversible
Hematogenous spread/Viremia
Virus reaches bloodstream via lymphatics or direct innoculation and replicates in endothelial cells or subepithelial blood vessels
Passive viremia
direct innoculation
Active viremia, primary
progeny viruses released from initial site of infection into the blood stream
Active viremia, secondary
Release of the virus from a localized area of secondary multiplication of the virus in the bloodstream
Cell associated viremia
viruses not cleared by Ab and tend to cause prolonged viremia
How can viruses be transported via neural spread?
axons
endoneural space
perineural lymphatics
infected Schwann cells
Centripedal movement
towards the brain
Centrifugal movement
CNS to body
Macule
flattened, red and inflammed, virus replicating in the dermis noninfectious
Papule
raised and reddened, local inflammation, noninfectious
Vesicle
fluid filled and raised, contains infectious virions
Pustule
Pus filled vesicle, can lead to scar formation
Infection of the GIT causes
enteritis, diarrhea, usually infect undifferentiated cell of villi
rapid destruction of enterocytes then replacement by cubiodal cells (self limiting infections)
Viral infectious of Resp tract can cause
Ciliostasis
Loss of mucus layer
Destruction of epithelial cells
Depressed alvelor macrophages
Neurotropic viruses cause
neuronal necrosis
nonsupprative encephalitis
perivascular infiltration of mononuclear cells (perivascular cuffing)
Infection of vascular epithelium leads to
activation of intrinisic pathway of clotting
edema and petechial hemorrhage
Disseminated Intravascular Coagulation
T/F Most viral infections of the dam have no effect on the fetus
True
Inapparent Viral Infection
Doesn’t cause noticeable illness
Most viruses are this
Acute viral infection followed by clearance
Short clinical course, virus cleared by host immune system
Persistant infections
Host immune response can’t clear the virus
carrier animals can continouly or intermitently shed the virus
Persistant infection type I
virus persists in a non infective form with intermitent periods of reactivation and shedding
Persistant Infection Type II
Chronic infection
Virus continually shed
host immune system can’t eliminate the virus
Persistant Infections Type 3
Slow infections, prolonged incubation periods
slow, progressive lethal dz (think prions)
Cytopathic effect
Virus damage/death to host cell
Inclusion bodies
Intracellular structures produced in cells by some viruses
Proto-oncogenes
Code for proteins that control normal cell growth
Oncogenes
genes coding for transformation of normal cells
Tumor suppresor genes
genes that code for proteins that control cell proliferation, arrest in G1
Rb protein
stops entry into S phase
unphosphorylated Rb protein
active- stops replication
phosphorylated Rb protein
inactivates- replication
p53
arrests cell cycle and apoptosis when cell is damaged
Oncogenic viruses
Mostly DNA viruses
Usually integrate into host genome
Lysins
hydrolytic enzymes that cleave cell walls
Retroviral integrase
enables retrovirus genome to be integrated into host DNA
Reverse transcriptase
generate cDNA from RNA template
Nucleic acid polymerase
viral genome replication
Neuraminidases
cleaves glycosidic bonds allowing release of viruses from host cells
Antigenic drift
point mutations within genes that code for antibody binding sites
Silent mutation
no change in a.a.
nonesense mutation
stop codon
missense mutation
change in aa
Genetic recombination
exchange of nucleotide sequences between 2 viruses
Genetic reassortment
Exchange of genome segments b/w related
viruses (happens faster than recombination)
Defective interfering mutants
deletion mutation, lacks one or more functional genes needed for virus replication
Need help of wild type virus, can be persistant
Viral interference
multiplication of virus is inhibited by an infective virus already in the cell
Pseudotype
related viruses infect the same cell and they swap genomes in their capsids
Pseudovirion
capsid encloses host nucleic acid, look like normal viruses but cannot replicate
What is a primary monolayer?
Cells taken directly from humans/animals
Heterogenous/Same chromosome #
Max 20 subcultures
What is a diploid cell line?
single cell type derived from human embryonic tissue or subcultures of a primary culture.
Can be subcultured 100 timesq
What is a continuous/immortal cell line?
capable of indefinite propagation in vitro
derived from tumor cells or by mutating primary or diploid cell lines
Do not resemble cells of origin, abnormal chromosome #
FDA prohibits their use in vaccine production
How do you prepare a monolayer?
- Treat tissue with a protease like trypsin
- Put in polystyrene container allowing anchorage
dependent growth and add growth medium - Incubate at optimal temperature for that species
How do you prepare from Rapid Cell Culture System
Rapid Cell Culture System
1.Grow a monolayer on a cover slip of a shell
vial [1-dram]
2.Add the clinical sample to the monolayer and centrifuge
3.Stain the monolayer with horseradish peroxidase or
antibodies to detect the virus
What grows on CAM
herpes and pox viruses
What is the color change in Growth medium?
Phenol red turns orange with cell growth
Virus Titration
quantitative determination of viral activity
Virus Titer
# of infectious units/ml of sample lowest concentration of virus that still infects cells
Physical Quantification
measures the exact # of virus in sample TEM Flow cytometry (virus counter 2100) Hemagglutination assay- Ag conc PCR
Biological Assay- Monolayer Plaque Assay
Cells are incubated as a monolayer and infected with a virus
Cells are placed in agar and the virus will kill infected cells
Counter stain so cells are clear against a red or purple background
determine titer as plaque forming units/ml
Biological Assay- Pock Assay
Titration of viruses on the CAM of the chick embryo, pock forming units/ml
Biological Assay- Transformation Assay
Same as monolayer but for oncogenic visues
Viruses transform cells so that they lose contact inhibition, observe mound (focus) of cells
focus forming units
Quantal Assay
No number only presence or absence of infection
TCID50
tissue culture infectivity dose
ELD50
Embryo lethal dose
PD50
Paralytic dose
Permissive Cell
productive infection in cell, virus hijacks cell machinery to replicate
Non-permissive cell
non productive infection, virus can’t replicate
Multiplicity of infection
of infectious viruses inoculated per cell
Eclipse period
time b/w uncoating and appearance of first progeny virions inside cells
Latent period
time from uncoating to just prior to the first release of viruses outside cells (for enveloped viruses- eclipse=latent period)
Burst size
height of curve= # of virions released
Where do most DNA viruses replicate?
Nucleus
Where do most RNA viruses replicate?
cytoplasm
In virus replication, how to viruses attach to a host cell?
VAPs bind to receptors on host cells
Ways a virus can have trouble with attachment
Abs can bind to VAP
Virus doesn’t have VAP to fit cell receptors
Explain virus penetration via receptor mediated endocytosis
Virions move into clathrin coated pits which form vesicles and fuse with lysosomal vesicles to form endosomes
How are enveloped viruses released from the endosome in receptor mediated endocytosis?
acidic enviroment in endosome
How are naked viruses released from endosome in receptor mediated endocytosis?
virion surface proteins are activated at low pH causing lysis of endosome and release of nucleocapsid
Explain virus penetration via surface fusion
Only enveloped viruses do this, the viral envelope fuses with the cell membrane via fusion proteins and the nucleocapsid immediately enters the cytoplasm of the cell. Virions can promote fusion b/w adjacent cells= syncytium. Viral proteins stay on the host cell membrane and can be a target for ADCC and complement.
Explain virus penetration via pore mediated
non enveloped viruses inject their genome into the host cytoplasm by making a pore in the host cell membrane
Explain virus penetration via antibody mediated
Viruses infect wbc’s, Ab binds to the virus and WBC engulfs the virus resulting in infection
What is virus uncoating?
Seperation of viral nucleic acid from envelope/capsid
Point at which the virus loses infectivity
(Enveloped viruses begins simultaneously with penetration)
When in the virus replication cycle will the virus not be detected?
Eclipse period
What does mRNA need for transcription?
5’ cap for stabilization and alignment on ribosomes
Poly A tail for signal transport and binding to ribosome
Polycistronic translation of proteins
one gene codes for a polyprotein and with protease makes more than one functional protein
Monocistronic translation of proteins
one gene codes for one protein
What enzymes are required for replication of viral DNA
helicase, ssDNA binding proteins, DNA polymerase, DNA ligase
dsDNA what type of replication?
semiconservative
ssDNA what type of replication?
needs replicative intermediate
Retroviruses need ______ for replication
DNA intermediate
What % of virions are infectious?
1-10%
How are naked viruses released?
lysis
How are enveloped viruses released?
bud from plasma membrane, ER, golgi, or nuclear membranes
Type I Viral Spread
Extracellular
Most common
Type II Viral Spread
Intercellular
Spread through desmosomes after cell fusion
Usually persistent infections
Type III Viral Spread
Nuclear
Viral genome is integrated into host genome, replicated and passed on to daughter cells
How are mucous membranes defended?
IgA and virucidal proteins
What are some protective mechanisms of the GIT
HCl, proteolytic enzymes, bile salts
What is the most common portal of entry for viruses?
Respiratory tract, via aerosol (coughing/sneezing)
What defends the Respiratory tract from viral infection?
Mucocilliar apparatus and cooler temp of URT
Local spread
on epithelial surfaces at site of entry->infect neighboring cells
Subepithelial invasion and lymphatic spread
virus breaches the basement membrane and reaches lymph nodes
Hematogenous spread/Viremia
Virus reaches bloodstream, presence of virus in bloodstream
Passive viremia
direct inoculation into bloodstream
Active viremia (primary)
progeny viruses released from initial site of replication to blood stream
Active viremia (secondary)
release of virus from localized area of secondary multiplication of the virus in the bloodstream
Which causes a longer viremia, free in plasma or cell-associated?
Cell Associated
Free in plasma is neutralized by ab
How can viruses have neural spread?
Transported in axons endoneural space perineural lymphatics infected schwann cells
Centripedal movement
towards brain
Centrifugal movement
CNS to body
How are most viruses spread to the CNS?
Viremia
Rash
temporary skin eruption
Macule
flat and reddened, virus replicating in the dermis + host inflam respons, noninfectious
Papule
raised and reddened, local inflam, noninfectious
Vesicle
fluid filled and raised, infectious
Pustule
pus filled vesicle, neutrophils, can lead to ulceration/scab formation
Where do GIT viruses normally infect? What is the result?
ingested viruses normally infect undifferentiated cells of villi. Result in rapid destruction of enterocytes and replacement by immature cuboidal cells that are resistant to viral infection- self limiting infections.
Viral infection of resp tract can cause
ciliostasis
loss of mucus layer
destruction of epithelial cells
depressed alveoloar macrophages
Neurotopic viruses cause
neuronal necrosis
nonsuppurative encephalitis
perivascular infiltration of mononuclear cells (perivascular scuffing)
Infection of endothelium leads to
activation of intrinsic pathway of clotting
edema and petechial hemorrhage
DIC (disseminated intravascular coagulation)
T/F most infections of the dam have no harmful effect on the fetus
t
Inapparent viral infection
asymptomatic, doesn’t cause noticeable illness
most viruses fall under this category
Acute viral infection
followed by viral clearance
short clinical course, virus cleared by immune system
Persistant Infection Type 1
Latent infection
virus persists in a a noninfectious form with intermittent periods of viral reactivation and shedding
Viral genome maintained in the cell
Persistant Infection Type 2
Chronic infection
Virus continually shed
Immune system can’t eliminate the infection
Persistant Infection Type 3
Slow infection
prolonged incubation period
Slow progressive lethal dz
(think prions type dz)
Cytopathic effect
damage/death to host cell
usually secondary effect, host can’t keep up w/ metabolic needs
Noncytocidal viruses
don’t cause immeadiate host cell death
often cause persistant infections
Inclusion bodies
intracellular structures produced in cells infected by some viruses, can be pathognomonic
Proto-oncogenes
gens coding for proteins that control normal cell growth
Oncogene
gene coding for proteins that transform normal cells
Tumor suppressor genes
encode proteins that inhibit cell proliferation
holds cell at G1
Rb protein
stops entry into S phase
When phosphorylating Rb protein
inactivates it- replication
When unphosphorylating Rb protein
active- stops replication
p53 protein
arrests cell cycle and apoptosis when DNA is damaged
Oncogenic viruses
only dna viruses
integrate into host cell genome
permissive cells
allow viruses to complete its replication cycle
non-permissive
virus can’t replicate, viral proteins end up causing transformation of host cell
Acute transforming viruses v-onc +
host cell proto-oncogene incorporated into viral genome= defective virus, can’t replicate
Chronic transforming viruses v-onc -
viruses lack v-onc genes, weakly oncogenic, viral gene promoter/enhancer may be integrated into the host genome near a proto-oncogene- enhanced expression
What are some characteristics of transformed cells?
spindle shaped
lose contact inhibition
express tumor antigens in cell membrane (FOCMA)
Type 1 Interferons
no viral specificity
stable at pH 2
Stimulate production of MHC1 protems and proteosome proteins
IFNa and IFNb
IFN a secreted by
virus infected leukocytes (macrophages)
not host specific
IFN-b secreted by
virus infected fibroblasts and epithelial cells
host species specific
Type 2 IFN
IFN-y
labile at pH 2
Host specific
Enhances expression of MHC 1 and 2
IFN-y secreted by
stimulated T and NK cells
Type 3 IFNs
IFN gamma 1,2,3
immunoregulation
Internal viral ag stimulate ________response
cell mediated
Surface viral Ag stimulate _________response
humoral (ab) + cell mediated
Viruses provoke what immune cells?
lymphocytes and monocyte/macrophages
During what stage of virus infection are Ab effective?
extracellular stage
Ab virus neutralization
inhibits viral attachment, penetration or uncoating
Viral ab opsonization
coat virions w/ IgG- phagocytozed by macrophages
Ab against viruses can
clump viruses
activate complement
ADCC
What is the major mechanism for virus infected cell destruction?
Cytotoxic T cells, recognize MHC1 proteins w/ viral Ag
Antigen multiplicity
ag w/ little-no cross reactivity
Antigen plasticity
rapidly changing surface antigens
T/F Most RNA viruses replicate w/i the cytoplasm of infected host cells
true
t/f ether decontaminated naked viruses
false
ether breaks down lipid but not capsid
Which viruses have a double layered capsid?
Reoviruses
According to Baltimore classification, DNA +/- without reverse transcriptase is
Class I
According to the Balitmore System, DNA + w/o reverse transcriptase is
Class II
According to the Baltimore Sytems, RNA +/- w/o reverse transcriptase is
Class III
According the Baltimore system, RNA + w/o reverse transcriptase
Class IV
According the Baltimore system, RNA - w/o reverse transcriptase
Class V
According the Baltimore system, RNA + w/ reverse transcriptase
Class VI
According the Baltimore system, DNA +/- w/ reverse transcriptase
Class VII
ICTV System suffix for Order
virales
ICTV System suffix for Family
viridae
ICTV System suffix for Subfamily
viridae
ICTV System suffix for Genus
virus
What is Rate Zonal Centrifugation
sample is layered on a density gradient (sucrose). Particles of different sizes sediment as discrete bands
What is Isopycnic Centrifugation
Particles are seperated by Buoyant Density (object has same density of fluid)
What is the Isopycnic point?
point at which the buoyant density of a partcle=surrounding density of gradient medium (sucrose of cesium chloride)
What is high performance liquid chromotography
quantification through UV
What is single radial immunodiffusion?
radial diffusion of purified viral ag and viral particles through agarose gel s/ polyclonal antisera against viral ag
How do you determine the titer
avg plaque count x reciprocal of the dilution selected
Transformation assay
quantification of oncogenic viruses, focus forming units
How do you calculate the TCID using Reed Munch eq
% infection above 50%-50%/infection above 50%-infection below 50%
Add the answer to the dilution next above 50%
Titer = answer/0.1mL
Routes of innoculation in eggs
o Yolk Sac
o Allantoic Cavity
o Amniotic Cavity
o Chorioallantoic Membrane
Typical ELISA
Ag in well, Ab tagged with enzyme, Ag binds to enzyme tagged Ab, wash excess unbound Ab, add substrate, enzyme tagged to Ab will change color of substrate. More color=more positive rxn
Direct ELISA
Ag immobilized, enzyme conjugated primary ab are used to detect/quantify ag conc. Specificity of primary Ab important.
Indirect ELISA
o Primary Ab not labeled, but detected instead w/ enzyme conjugated secondary Ab that recognize the primary Ab.
Sandwich ELISA
Ag bound between a layer of capture Ab and a layer of detection Ab.
Competitive ELISA
Ag of interest and Purified immobilized Ag compete from binding to the capture Ab. A decrease in signal w/ purified Ag alone indicated presence of Ag in sample.
Direct Flurescence Ab test
labeled Ab are added to Ag. Fluorescence appears at binding sites.
Indirect Flurescence Ab test
secondary Ab labeld with fluorescent marker that recognizes the primary antibody bound to ag.
Immunohistochemistry
Ab tagged w/ enzyme (horserasish peroxidase). Enzyme reacts w a substrate to produce a color you can see w/ a light microscope.
o Direct- Primary enzyme binds to Ag
o Indirect- Enzyme bound to secondary Ab that is specific against primary Ab.
Immunochromotography
o Point of care test, can be done in hospital. Ex- pregnancy test. Control band binds anti-antibodies
Complement fixation test
serum w/ ab binds to ag- no lysis is positive. RBCs lysed is neg
Hemadsorption inhibition test
Glycoproteins are inserted into host cell membrane at sites of budding enveloped viruses. Monolayer cells adsorb RBCs on their cell membranes. Ab bind to glycoproteins, wash excess Ab, incubate monolayer with RBCs, RBC binding is inhibited.
When is the best time to collect a sample from a patient
at the onset of symptoms
