UCBE

Virology Spring 2017

Study Guide: Virus Infection of Host Cells Ch 2 and 12

1. Concept of cytopathic effect
a. CPE is morphological cell damage due to viral replication and spread from one
cell to an adjacent or distant cell
b. CPE at cellular level leads to clinical symptoms of viral infection like fever, aches
and tissue damage
c. CPE is the direct outcome of the perturbation of host cells cellular metabolic
physiologic function and induction of cell death
d. CPE is visible without staining using low powered microscopes for morphological
alterations
i. Pyknosis: shrinkage of nucleus (Polio)
ii. membrane fragmentation: cell gets rounded and detach (Polio, adeno,
herpes)
iii. loss of contact inhibition: cells pile on each other (HPV and EBV)
iv. cell lysis (cytocidal virus): Replicates within cell and when cell cycle is
complete viral particles lyse out (Polio)
v. synctia formation (as in murine leukemia virus as well as in respiratory
syncytial virus). Glycoprotein of infected cell bind to receptors on
neighboring cell fusing
2. Features of CPE as discussed (check notes).
a. CPE in rabies, herpes, polio, reovirus, SV -40 and murine leukemia virus
infection.
i. Membrane proliferation
1. Common in polio and herpes virus
2. This is where the membrane serves as a scaffold where
replication occurs
ii. Fragmentation of organelles and chromosomes
1. Observed in herpes virus
2. Fragmentation because it is a DNA virus and wants to recycle
DNA from hsot
3. Envelopement in nucleus, denvelopment and renvelopement in
golgi
iii. Vacuoles in cytoplasm
1. Polyoma virus (SV-40) and Papilloma virus (HPV)
iv. Inclusions and inclusion bodies: nature and location is unique to
particular virus infection; facilitates laboratory diagnosis
1. Inclusions may represent
a. Accumulated viral components
i. Virion in nucleus: adeno virus and herpes virus
both are DNA viruses
ii. Virion in cytoplasm: Rabies virus and that is
clinically called negri bodies
iii. Factories in the cytoplasm which is observed in
pox and is manifested as guraineri
b. Altered host cell structures by infecting virus
 i. Reovirus: crescent shaped perinuclear inclusion
made of associated virion associated
microtubules
b. Adeno and herpes virus assembly in nucleus ( fig 13.8 and 13.5; no specific
details ).
i. Remodeling and resculpturing of nucleus
ii. Nucleus enlarges containing both mature as well as assembling virus
particles with infection progression
c. Viroplasm concept and specific makeup in rotavirus infection. Be clear on the
cellular components of viroplasm and consequence of adding inhibitors. Does it
contain viral ss or ds genome ? DS RNA GENOME
i. Viroplasms are discrete dynamic structures and assemblies formed in
cytoplasm and they are sites of viral replication and assembly
1. Viroplasms observed in rotavirus replication
a. 7 viral proteins
b. ds RNA genome segments
c. mRNA
d. cellular proteins and lipids that form platforms
i. perlipin from fat droplets
e. Genome mutations or infected cells exposed to lipid
droplet formation inhibitors leads to lower yield of
infectious virus particles and virus induced cell death
3. CPE timing in some viruses ( Herpes and Rubella only). Significance of CPE ?
a. CPE aids in monitoring progression of infection laboratory
b. Mutant viruses are characterized by their CPE (phenotypic trait)
c. CPE timing varies and is dependent on inoculum size
i. Herpes simplex virus observed in 1-2 days and destroys monolayers in 3
days
ii. CMV and Rubella virus takes several weeks
iii. Shell vial technique
1. Based on combination of cell culture, CPE, and immunologic
techniques
2. Shorts detection time from 6 days to 1-2 days
4. Identify / recall 4 -5 mechanisms of direct cell injury following virus infection. How they
are advantageous to the virus ? Should be able to identify at least 2 advantages as
discussed in class.
a. Cell injury at molecular level could be due to
i. Direct effect of viral entry and replication on host
1. Shut down nucleic acid synthesis
2. Shut down protein synthesis
3. Alter protein protein interaction
4. Altered vesicular transport
5. Altered cellular transcription activity
6. Advantages from these are
a. Cellular resources are easily available in the absence of
competing host cell components
b. Competition for translational machinery is virtually
eliminated between the viral mRNA and cellular mRNA
 ii. Affect of hosts intrinsic or adaptive immune response (skin rashes,
aches, fever)
5. How viral infection alters cell physiology? Significance of leaky membrane in RNA
stability.
a. Virus infection alters cell physiology because of de novo viral proteins and virus
interaction with cell membrane, which includes
i. Movement of ions and leaky membrane
1. Example is poliovirus infection which causes the initiation of
leaky membrane that results in an ion concentration that favors
viral mRNA translation over stable cellular mRNA
ii. Generation of second messengers: Leads to activation of cascade
pathway with altered cellular physiology
b. Should be clear on rotavirus nsP4 ( non structural protein 4 ) and its action as
enterotoxin ( mechanism of action).
i. Viral gene products alter cell physiology directly by
1. Adding purified viral proteins to cultured cells
2. Transfecting cell with viral gene in expression vector and de
novo protein synthesis
3. Example: Rotavirus nsP4 (non envelope protein, non capsid
protein) non structural protein 4a glycoprotein involved in
envelope formation
4. Cultured cells are transfected with rotavirus nsP4 inducing
phospholipase C dependent Ca++ signaling pathway leading to
Ca++ dependent Cl- secretion
5. Water molecules follow the electrolytes which causes diarrhea
creating a hypertonic environment
6. nsP4 acts as viral enterotoxin in intestinal mucosal cells
c. Consequence of inhibiting / blocking sodium glucose cotransporter ?
i. Ans : creates a typical hyperosmotic environment; subsequently water
is continuously drained out from the intestinal epithelial cells ).
Additionally secretion of chloride amplifies hyperosmosis resulting in
diarrhea associated with rotavirus infection.
6. Alteration of cellular biochemistry in viral infection.
a. Virus binding to cell surface sets in motion sequence of biochemical changes
that optimizes the intracellular environment for
i. Cellular synthetic machinery
ii. Productive infection using low molecular weight precurs
iii. Makes interior conducive to achieve goal for latency, chronic, slow, or
transforming infection
b. How fatty acid oxidation is altered by some viruses ( Hep B, C and herpesviruses)
upon infection of host cells.
i. Dengue, hepatitis B and C, herpes virus, and HIV-1 increase the
concentration of fatty acid synthase or their mRNA
ii. Patients with hepatitis B or C exhibit symptoms of steatosis (fat
accumulation in the liver), obesity, hepatocellular carcinoma which are
attributed to disturbance of lipid metabolism
c. Should be clear regarding HCMV and altered path of fatty acid synthesis. Role of
acetyl co A carboxylase and fatty acid synthase in viral infected cells?
 i. HCMV induces long chain fatty acid synthesis for infectious virus particle
ii. HCMV increases C flux from glucose (by 20 factor) to acetyl coA which
gets shuttled to the cytoplasm by citrate and then converted to malonyl
CoA a precursor for fatty acid synthase
iii. Inhibition of acetyl coA carboxylase or fatty acid synthase reduces yield
of infectious virus particles
d. Why lipid metabolism is altered in viral infected cells?
i. Viral envelop as well as membrane bound components impose an
increased demand for lipid synthesis
ii. HCMV
1. Increased fatty acids produced in infected cells
2. Nature in relation to C chain length
a. 10 fold increase in very long chain fatty acids
concentrated in infected cells
e. Specific lipids changes in HCV infection of host.
f. Altered lipid metabolism are common with enveloped / non-enveloped or in
both group of viruses?
i. Infection by non-enveloped viruses can also disturb lipid metabolism of
infected cells
ii. Cells infected with positive strand RNA viruses induce formation of
cytoplasmic membranous structure sites of viral genome replication and
assembly which may require reshaping the lipid storage depots
g. Polio infection and phosphatidyl choline synthesis.
i. Polio virus infection stimulates increased import of fatty acids initiated
by viral encoded proteins that leads to phospholipid synthesis
(phosphatidyl choline) within short time
ii. Increased activity of long chain acyl coA synthase is observed with
enhanced incorporation of long acyl chains (C16/18)
iii. inhibiting production of acyl synthase by RNA interference assay impairs
poliovirus replication
7. Whats the significance of intracellular alteration of host genes?
a. Altered gene expression of host cells may include
i. Inhibition of cellular mRNA synthesis (cellular transcription process
dampens)
ii. Inhibtion of cellular mRNA translation
b. With infection progression, there is differential expression of certain genes, it
can be up or down regulated
i. Some biochemical function may be upregulated to enhance virus
replication
c. Role of transactivators in viral replication cycle.
i. Some viral genome encodes powerful transcription activators like SP-40
Large T antigen
1. Promotes viral gene expression, influencing cellular mRNA
synthesis too during the process
d. Do viral DNAs have mammalian transcription factors binding site? Whats the
role of these sites? And how host cells factors are activated?
i. Viral DNA contains sequence motifs for binding diverse mammalian
transcription factors
 ii. These in conjunction with viral coded regulatory proteins are involved in
activation and repression of viral or cellular genes
iii. These sites are critical for establishment of latent or transforming viral
infection or to produce progeny virus
e. Role of transcription factors of host cell?
i. Viral gene expression regulation is a basic mechanism similar to host cell
ii. Virus cell receptor interaction initiates signaling cascade that leads to
activation of transcription factors followed by binding to viral DNA
sequence
iii. The transcription factors include:
1. NFKB (kappa beta), CREB 1, OCT 1, AP 1, NFAT (nuclear factor
of activated T cell)
2. These are activated by the virus binding to the cell surface
receptor
3. The activation process may include: phosphorylation
dephosphorylation, dimerization, dissociation from inhibitory
subunits
f. Advantages of inhibiting host mRNA production by RNA viruses. Recall /identify
2 ways cellular mRNA synthesis can be blocked by viruses.
i. Viral genes may encode proteins that inhibit cellular gene expression
1. Dampening and blocking cellular mechanisms that triggers
antiviral responses or modulating host signal transduction
pathways favors viral survival
ii. Viruses with positive or negative RNA genomes inhibit preferentially
host cells mRNA production because
1. Viral genome expression has minimal dependence on cellular
system
2. Promotes selective and efficient synthesis of viral proteins due
to minimal or no competition for translation machinery
3. Eliminates triggering of antiviral response
g. How polio and alpha virus coded protein inhibits host cells mRNA synthesis
i. Viral protein targets for inhibition or disruption are
1. Cellular proteins like TATA binding proteins that are involved in
cellular mRNA synthesis in nucleus
2. Proteins necessary for splicing or export to cytoplasm
3. Block translation of cellular mRNA (polio virus)
a. Polio 3Cpro targets TBP (shuts down transcriptional
machinery); poliovirus with short infectious cycle
demands very drastic measures to prevent cellular
protein synthesis
b. Alphavirus NS2 targets RNA poll II (catalytic subunit)
i. Alpha viruses are RNA viruses that shut down
cellular RNA polymerase II
8. Virus isolation from clinical samples as discussed in class. Stabilizing proteins commonly
used.
a. There should be a short time between specimen collection and delivery to lab
because there is a greater possibility for virus isolation, reasons include:
 i. Many viruses are fragile, which is true for many pathogenic viruses
(some are enveloped)
ii. Sample could be susceptible to bacterial and fungal overgrowth which
can impact or interfere with viral sample
b. It is best to transport in ice using special media containing antibiotics and
proteins (bovine serum albumin, gelatin)
i. With enveloped viruses, there is increased risk for titer loss if it is kept
at room temperature or frozen at -20 C
ii. Not a problem with non-enveloped viruses because they are more
resistant to temperature
1. Example: Entero and adenovirus
9. Identify 3 main ways of virus cultivation. Why viruses cant be grown in culture medium
unlike bacterial cells? Limitations of tissue culture for viral cultivation.
a. Tissue culture is routinely used to study animal viruses because viruses demand
living cells to support their replication
i. Culture medium are used for bacterial cell growth, but for viruses you
cant use the culture medium because they require living cells
b. Limitation in culture tissue:
i. Obtaining a large number of animal cells
ii. Expensive and technically demanding
c. Phages are more extensively studied because bacterial cells are easy to grow
and they are simpler than eukaryotic cells
d. John Enders, Thomas Weller, and Fredrick Robbins were awarded Noble prize in
1954 because
i. First to report that PV could multiply in non neural cultured cells in
human embryonic skin and muscle, laying the foundation for:
1. Propagation of ther viruses
2. Discovery of new viruses
3. Viral vaccine development
ii. Synchronous infection is when all the cells in a medium are infected
with a virus and all cells follow the same pathway of infection
iii. Synchronous infections provided the ability to study molecular biology
and biochemistry of viral replication (valuable)
e. The three main ways of virus cultivation are
i. Whole organisms
ii. Cell/tissue culture
iii. Embryonated eggs
10. Should be able to recall / identify the advantages and disadvantages using whole
animals in virus cultivation. Are they used in clinical labs? Diversity in use of whole
organism for virus cultivation.
a. Whole organism advantages
i. Useful for studying pathogenesis of viral infection
1. Examples HIV SIV
ii. Used for vaccine development and to evaluate vaccine safety
b. Disadvantage
i. Animals with pathogenic viruses are expensive to bread and maintain
ii. Complexity makes interpreting results difficult; host variation often
leads to non-reproducible results
 iii. Wasteful use in research is morally unacceptable
c. Exptl animals are rarely used in clinical labs due to above reasons; normally use
cell/tissue culture
11. Cell/Tissue Culture: Three types are routinely used for virus cultivation and they are
expensive and demanding.
a. Three types of cell/tissue culture are: Primary cell culture, Continuous cell
culture, suspension culture
b. The gold standard for viral isolation and detection etc. Primary cell culture and
its feature. Be clear when PCC are used (recall at least one). Continuous cell
culture.
i. Primary cell culture (PCC): traditionally regarded as gold standard for
detection, isolation and diagnosis of viruses
1. Obtained directly from tissue slices
2. Cells have limited life span (5-20 cell division)
3. PCC can be heterogeneous or homogenous; diploid cell line is
preferred over haploid
a. If two or more different types in the cell it is
heterogeneous diploid
4. Diploid cell line and its use
a. For a viral vaccine it is mandatory to use a diploid PCC
(FDA regulation)
i. Commonly used PCC in virus study:
1. Human embryonic lung cells, primary
human foreskin fibroblasts, kidney cells
of human and monkey, embryonic cell
lines of mouse and chicken
ii. Chicken and mouse embryonic cell lines; their
use in experimental virology, unavailability of
appropriate cell lines and when cell
differentiation state is unimportant
1. Embryonic cell lines of mouse and
chicken only used in experimental
virology when appropriate cell lines are
unavailable and the state of cell
differentiation is important, meaning
that when cells have aged there will be
phenotypic changes
c. How continuous cell culture is different from primary culture. Are continuous
culture used for vaccine prep? Identify 3T3 mouse fibroblasts (NIH3T3 cell line)
as well as HeLa and Vero cells belongs to which group (primary or continuous
cell lines?). How can one obtain continuous cell line from PCC? Uniqueness of
Hela cells (cervical cancer cells and are no more diploid since the cells have lost
many chromosomes)
i. Continuous cell culture: never used for viral vaccine cultivation
1. Single type cells (homogenous) immortalized and easy to
maintain
 2. Derived from tumor tissue or treating primary cells lines;
infected with tumorigenic virus or irradiated or treated with
mutagenic agent
3. Can be synchronously infected for one step growth curve
4. Commonly used lines are:
a. Hela cell cervical cancer cell; no more diploid cell
because may have lost chromosome and have become
haploid
b. Vero cell line afrivan green monkey;
c. NIH 3T3 mouse fibroblast cell line
d. Suspension culture and micro beads use in virus isolation? Why Hela cells cant
be used for virus cultivation in vaccine preparations?
i. Can be of primary or continuous cell lines
ii. Cells are bathed in a nutrient enriched medium
iii. A magnet continuously stirs the medium
iv. Advantage is the large number of cells that can be infected in small
volume; This is useful for virus isolation in large amount
v. Lately microbeads are used, which provides greater surface area for
cells; microbeads provides surface for cells to attach
BIOLOGICAL ASSAY
12. Should be able to identify some advantages / disadvantages of using lab animals and
eggs to cultivate viruses. Pocks in egg membrane and vaccinia virus as 1st quantitative
assay for virus.
a. Embryonated eggs
i. The developing embryo provides a system to evaluate how the body
reacts to a virus
ii. Used to cultivate influenza and strains of vaccinia virus when large
volume of virus antigen is needed
1. First quantitative assay for a virus was done for vaccinia virus by
counting the pocks number on the chorioallantoic membrane
2. 5-14 days post fertilization, hole is drilled, virus injected
b. Chicken egg is useful because
i. Inexpensive
ii. Large cell
iii. Free of contaminating microbes because the farm makes sure they are
free of contamination (like salmonella)
iv. Nourishing yolk (makes itself sufficient)
13. Advantage and disadvantages of infectivity assay. Its effectiveness for virus that forms
plaques?
a. Infectivity assay
i. Disadvantage: slow, tedious, time consuming, and not available for
some viruses
ii. Norwalk virus infectivity assay: need some factors supplied by bacteria;
Norwalk is supplied by Enterobacter cloacae (intestinal bacteria that
express histo blood group antigens which are needed B cells infection)
14. Explain how antibodies or viral antigens use aids virus detection.
a. Serological Assay
i. Use of specific antibodies or viral antigen, this is useful since it can
 1. Viral antigens like HIV or Hepatitis B surface antigen
ii. Detect presence of viral proteins in infected cells or tissues
iii. Confirm antibodies in host, a result or outcome of virus infection
15. Molecular methods (PCR, RT PCR etc): its use / advantage. Why its useful to detect HPV
using molecular method and not by Abs use.
a. Molecular methods
i. Detection of viral nucleic acid sequences
1. PCR techniques or DNA probes and in situ hybridization
ii. Sensitive and specific like antibodies; detects slow replicating or non
productive viruses
1. No CPE
2. benign virus have no CPE but can still detect them
iii. Useful when viral antigen cant be detected using antibodies
16. What is plaque, virus titer and plaque purification? Usefulness of plaque assay. Why its
overlayered by soft agar? Why healthy, viable cells are used for successful plaque assay.
a. Biological: Virus Infectivity Assay
b. Plaque assay
i. confirms infectivity, reliable process and easy to perform
ii. useful for viruses that infects monolayer cells and forms visible plaques
(also limitation)
iii. Prerequisite: Cells should be healthy with 95% viability at time of assay;
no CPE then plaque assay doesnt work
iv. Virus Titre: refers to concentration of virus in sample (relative amount):
calculated as pfu/ml
1. Used for
a. Obtaining virus titer: quantitate viral stock
b. Testing effectiveness of antiviral drugs
i. Only cytoltic viruses are quantified with this
assay
ii. Accurate when low virus dilutions are used
otherwise difficult to quantitate plaques; if it
isnt low there will be too much to count
iii. Overlayered by soft agar; dye is used to
enhance contrast
1. Dead cells cant pick up dye so you will
see area (plaque) in the background;
living cells pick up and retain the dye
iv. Plaques counted
1. You have a cultured medium and do a
serial dilution, after a couple dilution
you take out .1mL and do a plaque
assay; too many to count (TMTC) or too
little to count plates cant be used; you
want 20-50 depending on the plate
v. A single virion makes a plaque: your presume that one virus infected the
cell and killed neighboring cells
vi. Plaque purified: virus stock is prepared from a single plaque, the virus is
sucked out of the area
 c. Concept of fluorescent and transformation assays for virus detection with
examples as discussed.
i. How are noncytolytic virus quantitated? Identify if these 2 methods are
based upon induction of cell death?
1. Fluorescent focus assay
a. Used for noncytolytic viruses; Not dependent on the
ability of viruse to induce cell death
b. Uses antibody staining method to detect viral infected
cells as early as two days much earlier than other
methods
c. Why cells needs to be permeabilized by Triton X 100 or
cold acetone; alcohol?
i. Cells need to be permeabilized so the antibody
can enter the cell following adsorption; Cells are
permeabilized by using 0.01-0.2% triton X100 or
cold acetone: alcohol mixture
ii. Initially incubated with primary antibody
against viral protein
iii. Second antibody with fluorescent tag, which
recognizes primary antibody is added; unbound
antibody is washed
iv. Cells examined fluorescent microscope
d. Titer: expressed as fluorescent focus forming unit/ml
ii. RSV and transformation assay use.
1. Transformation assays
a. Useful for viruses that dont form plaques (noncytolytic)
b. Transformed cells loose contact inhibition and become
anchorage independent; will try to pile on top of each
forming distinct foci and become anchorage
independent
c. Cells pile on each other forming distinct foci (compare
to the monolayer elsewhere)
d. Titer: expressed in focus forming units/ml
i. Example: Rous Sarcoma virus transforms
chicken embryo cells
PHYSICAL ASSAYS: some are rapid and easy to carry out and some are harder to perform
17. Electron microscope detection of viruses.
a. Electron microscopy
i. Virus particles are visualized in specimen; provides idea of quantitative
measurement, before this they to actually look at poop
ii. Limitations and disadvantages of EM. Provide a virus name that is
directly detected using EM from clinical sample.
1. Advantages
a. Good for viruses that cant be cultured or no tissue
culture system has been developed or exists
b. Structural details can be observed clearly and evaluated
if stains or probes penetrate any structure on virus
surface
 2. Disadvantages
a. Microscope expensive
b. Demands experienced personal
i. This is why we look for antibodies in HIV; due to
the tedious processing to get a blood sample
and examine via electron microscope
c. Though specific for many viruses processing is tedious
i. Norwalk cultivation (and the role of E cloacae)
and identification of SARS Co V using
conventional virologic techniques. Do we have a
method to cultivate Norwalk virus available
now? More details to follow in new slide
ii. Human coronavirus (SARS) was isolated or
detected using classical or conventional virology
techniques
1. Tissue culture, microscopes, etc.
2. Dont underestimate classical methods
b. Measuring Enzyme activity: polymerase assay
i. Permeabilized cells or particles and radioactive precursor is used
ii. Radioactivity incorporated in nucleic acid is quantitated
iii. Used for viruses that dont form plaques or transform cells, such as
retroviruses
iv. Allows investigators a quick estimate of virus production during course
of an infection
18. Hemagglutination assay and the basis of its working. Can all virus be detected using this
assay?
a. Hemagglutination assay
i. Indirect method to quantify virus particles
ii. Exhibited by some viruses to clump RBCs because of hemagglutinin
spike proteins
1. Binds to N acetyl neuraminic acid (sialic acid of RBCs
glycoprotein) on RBCs
iii. Unabsorbed RBCs settle at bottom of the well forming short dot
iv. Agglutinated RBCs forms a diffuse pattern coating the well; prevented
from settling out of suspension
v. Serially diluted virus prep is added to fixed amount of RBC
vi. Why RBC assay shouldnt be incubated with virus for more than 30
mins?
1. Assay shouldnt be incubated for more than 30 minutes because
it will give false results
vii. Not sensitive to determine small number of virus particle
viii. Why RBC needs to be fresh? Does this method provide any insight /
information about replication of virus?
1. RBC has to be freshly prepared or isolated because
agglutination activity promptly declines over time and also this
process doesnt provide or shed any light on viral replication
since it is not a requirement for hemagglutination
19. Concept of plaque to PFU ratio. Recall / identify the reasons for wide range of the ratio
in animal virus. Pseudovirion and VLPs concept (as it relates to HPV only)
a. Particle to PFU Ratio
i. Is the ratio of physical particles in sample or infectious particles
ii. Assumptions: intact virus particles are infectious
iii. Provides idea of physical intact infectious particle in viral preparation
iv. Same virus preparation
1. Plaque assay: parameter for number of infectious virus particles
2. Electron microscopy: quantitative measure of number of
physical virus particles
v. Bacteriophage: ratio is 1
1. Animal virus: variable wide range from 1-1000 indicates that not
all viruses are infectious (complicates issues)
2. Many reasons for the high ratio
a. Particle intactness: loss or disruption of envelope or
altered viral surface protein which may lead to
inefficient attachment
b. Particles with defective genomes (non infectious
particles) in extreme case higher proportion may be
defective particles
i. Frequent occurrence of mutations, deletions,
etc.
c. Capsid assembly with no viral genome
i. Incorporate cellular DNA or RNA instead of viral
genome (pseudovirions aka Virus Like Particles)
normally, packaging signals ensures viral nucleic
acid in capsids
d. Host cell may have antiviral defense mechanism;
preparation may contain wild type intact virion but host
cell intrinsic defense mechanism overrides replication
mechanism resulting in no progeny virion produced
despite viral entry
i. Host cell defense system overrides it and
prevents the outcome of viral progeny even if
everything is packaged
e. Complexity of infectious cycle; many competent virions
fails
20. Serological methods: detection and diagnosis. Compare 1st Ab vs 2nd Ab response
against virus particle.
a. Serological Methods in Virion Infection: Detection and Diagnosis
i. Antibodies developed against key viral antigens or proteins can identify
viral disease or in some, slow course of viral infection
a. Which Abs is specific for viral internal protein / structural protein?
a. 1st antibodies are directed toward viral antigens that are freely available
or accessible to immune system
i. expressed on virion surface (spike proteins or capsid proteins)
nd
b. 2 antibodies are directed against intracellular viral proteins or
enzymes
 i. these antibody levels rise during course of viral infection when
infecting viruses or cellular immune response has lysed cells
because T lymphocyte recognize some of the infected cells and
when they lyse we develop antibodies
21. Immunostaining: recall advantages associated with immunostaining. Its use in clinical
setting for RSV detection only (Influenza, measles and adenovirus FYI) For Information
only differences between direct and indirect immunostaining and the dyes
a. Immunostaining
i. Used for viral protein detection in infected cells or tissues using specific
antibodies
1. Cells fixed and permeablized before adding antibodies; cells are
fixed when they are stuck to cultured dish or flask
2. Antibodies tagged with indicators are used for visual detection
a. Fluorescent dyes
b. Enzymes
ii. Advantages
1. Probe viral replication site within host cell
2. Observe subcellular distribution of viral proteins in infected cell
3. Monitor viral translation kinetics inside cells (time course
analysis)
iii. Clinical usage: diagnosis of Respiratory Synctial Virus, influenza,
measles, and adenovirus
iv. Direct staining: indicator coupled to primary antibody
v. Indirect staining: the second antibody has coupled indicator, non-
specific signals are reduced or eliminated
22. Principle of immunoprecipitation and immunoblotting. Recall some advantages and
disadvantages of the 2 processes. For immunoprecipitation, disadvantage is use of
radioactive material (S35 methionine) to label viral proteins.
a. Immunoprecipitation
i. Based on specific interaction between viral antibody and protein
antigens in solubilized extracts of infected cells or tissues
ii. Provides information regarding viral proteins such as: molecular weight,
sub cellular localization, association with other viral or cellular proteins
iii. Can enrich proteins of interest to improve overall sensitivity for
detection
iv. Disadvantage
1. Use of radioactive material for protein labeling
2. You can label viral proteins using S35 methionine and all the
newly synthesized proteins will have S35 methionine which is a
disadvantage because it can give false positive results
b. Immunoblotting (Western Blotting)
i. Based on specific reactions between viral proteins on an inert
membrane and specific antibody
ii. Advantage
1. No radioactive labeling or use, instead antibodies are tagged
with sensitive indicator used, ideal for tissues organs or cultured
cells
iii. Disadvantage
 1. Doesnt provide clue for kinetics of viral protein synthesis
2. Fails to reveal if viral protein is associated with other protein
(cellular or viral get separated on SDS page)
3. Can produce non-specific or indeterminate signals
a. Indeterminate test results of HIV
iv. Most common usage is to detect HIV in donated blood and clinical
samples, also used for other virus detection
23. ELISA and its use. Concept of Shell Vial technique and its clinical use.
a. Enzyme Linked Immunosorbent Assay (ELISA) Detecting Viral Antibodies or
Antigens
i. Used for screening virus or presence of antibodies in serum samples
ii. Based upon antigen antibody reaction followed by detection via a
common antibody conjugated to an enzyme that produces a color
change
iii. Sensitive assay for HIV Detection
b. Centrifugation Culture (Shell vial Technique) use in clinical labs
i. Detects viral antigen before CPE are visible
1. Viral infection confirmed within 1-2 days
2. Increases efficiency: infection progression is enhanced
c. Lateral flow immunochromatographic assay as discussed in side and in class.
Recall some advantages of this method.
i. Lateral Flow Immunochromatographic Assay
1. Modification of EIA
a. Used in rapid antigen detection kits
b. Slide covered with membrane used to detect viral
antigen
c. Commercial kits are available for influenza virus,
respiratory syncytial virus, and rotavirus
2. Antibody conjugated to a detector and reacts with antigen in
clinical sample applied at the absorbent pad, the antibody
antigen complex migrates across the membrane and is captured
by a secondary antibody, resulting in a visible line that indicates
viral antigen presence
3. The advantage is no instrument is required and it can be read
within 5-20 minutes in a physicians office
24. Limitations of serological methods. Explain how parainfluenzae and mumps infection
may leads to false positive results. Monoclonal Ab use and false negative results with
some viruses. Hepatitis B infection and detection using Ab and immune complex:
limitations.
a. Serological Methods Limitations
i. Fails to indicate when infection had occurred or intiated
ii. False positive or negative result tends to confuse diagnosis
iii. Possible that individual antibodies may be in immune complex thereby
preventing detection
1. Hepatitis B antibodies bound to antigen, Hep B has many
particles which include filamentous and spherical and the
antibodies could bind to that and there will be no detection
 iv. Occasionally serological cross reaction between different viruses may
confuse identity of the agent (para influenza and mumps express
related antigens)
b. If antibody used is too specific, such as monoclonal antibody, may not recognize
different strains from same family; tends to give false negative results
c. Like HPV has 30 strains and humans can be infected by 15 different ones and
you use a monoclonal antibody for one strain you cant pick up the other strain;
same thing goes for rhinovirus
d. Being aware of the limitations of diagnostic methods along with good
knowledge of signs and symptoms aids viral infection diagnosis
25. Concept of eclipse and latent phase in growth curve. Should be able to recall some
specific features. Explain why infectious enveloped viruses cant be detected in latent
phase. Why moi beyond certain limit is of no use?
a. Virus growth curve (One Sept growth curve)
i. For studying single life cycle of virus, synchronous infection is the key
1. Main feature for successful growth curve is synchronous
infection because all cells are being infected at the same time
and have same steps and cycle
ii. Eclipse period (overshadowed; latent phase includes this phase)
1. Extends from entry through biosynthesis
2. Genome separates from capsid (uncoating)
3. Infectious particles not detected inside cells; components being
synthesized, processed and assembled
4. Doesnt include the attachment step; the eclipse phase begins
when the virus has physically entered the cell
5. Low infectivity due to: unadsorbed virus or adsorbed virus not
uncoated (results in short eclipse phase)
iii. Latent Phase
1. Spans from entry through release
2. Inclusive of eclipse period
3. Intracellular virus numbers starts to increase; onset of synthetic
phase
4. Plateaus eventually since host cell becomes metabolically
limited and cant support replication any more
b. Cells infected at high moi (multiplicity of infection) 10
i. MOI number of infectious virus particles added per susceptible cells
1. 10-100 moi used
ii. MOI beyond certain value doesnt yield new virus since host cells have
finite capacity to make new virus particles
c. Growth curve periods essential because
i. idea of virus yield: varies dependent upon whether it is a DNA or RNA
virus
ii. Information about system virus system is important to study mutant
viruses (mutant virus can be used as vaccine candidate)
iii. Idea regarding multiplication of a virus or analysis of a new virus host
system
d. Results of growth curve should be interpreted with caution, since it varies
widely
 i. Enveloped viruses released via budding becomes infectious only after
exit; so wild type intracellular infectious virus cant be detected because
they only become infective when they are released
ii. Small RNA viruses, entire growth curve is completed in 6-8 hours; the
passes are shorter like polio in 6 hours infected cells are lysed in 8 hours
all cells are lysed and new virus particles are made
iii. DNA viruses contrast have extended latent and synthesis phase because
it takes time to get into nucleus and compete with host DNA machinery
and to ensure that resources are available