midterm examSPECIMEN CULTIVATION FOR VIROLOGY Flashcards
The primary purposes of viral cultivation are
- To isolate and identify viruses in clinical specimens
- To prepare viruses for vaccines
- And to do detailed research on viral structure, multiplication
cycles, genetics, and effects on host cells
The earliest method for the cultivation of viruses causing
human diseases was inoculation into __
human volunteers.
they used human volunteers for
their pioneering work on yellow fever
Reed and colleagues (1900)
Due to serious risk involved, human volunteers are used
only when __
when no other method is available and when the virus
is relatively harmless
LABORATORY DIAGNOSIS OF VIRAL INFECTION
I. Identification of the virus in cell culture
II. Microscopic identification in the specimen
III. Serological procedures to detect a rise in antibody titer
IV. Detection of viral antigen in blood or body fluids
V. Detection of viral nucleic acids
METHODS OF VIRAL ISOLATION
- Animal inoculation
- embryonated egg inoculation
- tissue culture
this type of isolation is the primary isolation of certain viruses
animal inoculation
this type of isolation is for us to study the pathogenesis, immune response, and epidemiology of a viral disease
animal inoculation
this isolation method as well is used for the study of oncogenesis
animal inoculation
- they play
an essential role in studies
of viral pathogenesis
Laboratory animals
they used Monkeys for the
isolation of poliovirus
Landsteiner and Popper
(1909)
Landsteiner and Popper
(1909)-Monkeys for the
isolation of poliovirus
why is it risky?
potential to physical aggression and cause harm
he introduce white mice
Theiler (1903)
why monkey is not used anymore as a disease model?
they can’t exhibit human diseases
what type of mice is used in lab
Infant (Suckling mice)
suckling mice are susceptible to which viruses
Coxsackie and arbovirus
Routes of inoculation for animal inoculation
Intracerebral,
subcutaneous,
intraperitoneal,
intranasal
how do we confirm if there’s growth of virus inside the animal model
sign of Death, disease or visible
lesions
Disadvantages of animal inoculation
- costly
- maintenance
- interference of immune system
- individual variations (gender)
- difficulty in choosing of animals for particular virus
embryonated egg inoculation is started by whom
goodpasture (1931)
embryonated egg inoculation is started by goodpasture and is further developed by __
burnet
the embryonate egg we must use for inoculation is how many days old?
8-11 days old
the embryonated egg must be incubated for how many days
2-9 days
- Eggs provide a suitable
means for:
the primary isolation and identification of viruses
maintenance of stock cultures
and the production of vaccines
Routes of Inoculation of embryonated egg inoculation
- Chorioallantoic membrane(CAM)
2.Allantoic Cavity - Amniotic Cavity
- Yolk sac
in embryonated egg, viruses lies in ChorQioallantoic membrane(CAM)
Herpes simplex virus
poxvirus
rous sarcoma virus
in embryonated egg, viruses lies in amniotic cavity
influenza virus
mumps virus
in embryonated egg, viruses lies in yolk sac
herpes simplex virus
in embryonated egg, viruses lies in ALLANTOIC
influenza virus
mumps virus
newcastle disease virus
avian adenovirus
we look for ___ in Chorioallantoic Membrane
(CAM) to know if there’s growth of virus
pocks - visible lesion
most popular inoculation for embryonated egg
allantoic cavity
Has been widely used in
veterinary virology when it comes to embryonated egg
. Chorioallantoic Membrane
(CAM)
how do we interpret the result if it’s inoculated in allantoic cavity
fluid is examined for turbidity or hemagglutination
this type of inoculation for embryonated egg Has little application in
veterinary virology
amniotic cavity
Virus is introduced directly into
the amniotic fluid that bathes the
developing embryo
amniotic cavity
Volume of fluid in the
infected amniotic sac is small
(1-2 ml)
Simplest method for growth
and multiplication of virus in terms of embryonated egg
yolk sac
EMBRYONATED EGG ROUTES OF INOCULATION
Immune interference
mechanism can be detected
in most avian viruses
yolk sac
this inoculation method for embryonated egg Can also be used for the
cultivation of Chlamydia
and Rickettsia
yolk sac
Process of holding a strong light above
or below the egg to observe the
embryo
egg candling
Detection of Viral Growth in embryonated egg inoculation
death of the embryo
defect in embryonic development
localized areas of damage in the membranes (pocks)
A crucial technique in viral
isolation that involved
cultivating viruses in living
cells or tissues.
tissue culture
Provides a controlled
environment for studying
replication, pathogenesis
and development of
antiviral drugs and vaccines
tissue culture
TYPES OF TISSUE CULTURES
organ culture
explant culture
cell culture
a type of tissue culture wherein Small bits of organs can
be maintained in vitro
organ culture
a type of tissue culture wherein it’s useful for the isolation of some viruses which appear to be highly specialized parasites of certain organs
organ culture
a tissue culture wherein Fragments of minced
tissue can be grown as
‘explant’ embedded in
plasma clots
explant culture
a Routinely used type of tissue culture
cell culture
a type of tissue culture that is Dissociated using proteolytic
enzymes (trypsin) and
mechanical shaking
cell culture
the type of tissue culture in which the Growth medium contains essential amino acids,
vitamins, salts, glucose and a buffering system of bicarbonate in equilibrium
with atmosphere
containing 5% carbon
dioxide
cell culture
tissue culture specifically the cell culture, is Supplemented with up to
______ serum
5% calf or fetal calf
indicator used in cell culture
phenol red
CLASSIFICATION OF CELL CULTURES
- Primary Cell Cultures
- Diploid Cell Lines
- Continuous Cell Lines
the CLASSIFICATION OF CELL CULTURES is based on
origin
chromosomal characteristics
number of generations through which they can be maintained
this type of cell culture in which the Normal cells obtained from
fresh organs of animals or
human being and cultured
primary cell cultures
this type of cell culture is Capable of only limited growth
in culture and cannot be
maintained in serial culture (1-2
passage
primary cell culture
example of primary cell culture
- Monkey kidney cell culture.
- Human embryonic kidney.
- Chick embryo cell culture
this type of cell culture is Commonly employed for
primary isolation of viruses and
in preparation of vaccine
primary cell culture
a type of cell culture that is a Diploid cells and have complete
set of chromosomes
diploid cell lines
the type of cell culture - diploid cell lines has a * Limited lifespan ___
(20-50
serial passages)
Useful for isolation of some
FASTIDIOUS pathogens and
for the production of viral
vaccines
diploid cell lines
this type of cell culture which Cells of a single type, usually
derived from cancer cells, that are
capable of continuous serial
cultivation indefinitely
continuous cell lines
example of continuous cell lines
Hela, hep-2 and KB cell lines
continuous cell lines are Maintained by __
serial subcultivation
or stored in the cold (–70°C)
is continuous cell line Now permitted to be used for
vaccine manufacture,?
yes, eg. rabies vaxx
HeLa came from
Henrietta Lacks
Hep 2 came from
human larynx epidermal carcinoma.
Rapid modification of conventional
cell culture
shell vial cell culture
shell vial cell culture
involved culturing cells in a small,
round-bottomed vial referred to
as a “__”
shell vial
shell vial cell culture is Incubated for__hours
24 to 48
DETECTION OF VIRUS GROWTH IN CELL CULTURE
- Cytopathic effect
- Metabolic inhibition
- Hemadsorption
- Interference
- Transformation
- Immunofluorescence
- Detection of virus-specific nucleic acid
- Detection of enzymes
one of the way to detect virus growth in a cell culture
refer to the components in an infected cell or abnormal
accumulations of cellular materials resulting from virus-induced
metabolic disruption
viral inclusions
aggregates of cells fused to form one large cell
with multiple nuclei
syncytial cells
Morphological changes in cultured cells - ‘___’ (CPE) caused by
‘cytopathogenic viruses.’
cytopathic effects
Presumptive identification of a virus isolated from a clinical specimen.
cytopathic effect
Main Types of CPE
rounding of cells
cell necrosis and lysis
syncytium formation
discrete focal degeneration
rounding and aggregation
this virus causes a cytopathic effect such as rounding of cells
picornaviruses
this virus causes a cytopathic effect such as cell necrosis and lysis
enteroviruses
this virus causes a cytopathic effect such as syncytium formation
measles, respiratory syncytial virus, human immunodeficiency virus
this virus causes a cytopathic effect such as discrete focal degeneration
herpes virus
this virus causes a cytopathic effect such as rounding and aggregation
adenovirus
quantitation of cell culture
negative
uninfected monolayer
quantitation of cell culture
equivocal
atypical alteration of monolayer involving few cells
quantitation of cell culture
1+
1%-25% of monolayer exhibits cytopathic effects
quantitation of cell culture
2+
25%-50% of monolayer exhibits CPE
quantitation of cell culture
3+
50%-75% of monolayer exhibits CPE
quantitation of cell culture
4+
76% to 100 % of monolayer exhibits CPE
an indicator of growth of virus in cell culture
Viruses interfere with the metabolic activities of infected cells
leading to reduction in cellular metabolic process
metabolic inhibition
in metabolic inhibition, there will be changes in what parameter
pH
in metabolic inhibition, normal cell culture’s medium will turn to what pH
medium turns basic because the normal cell culture must be acidic. If there’s a growth of virus, the medium will be basic
virus in cell culture means ____ acid production in medium
no acid production causing the medium turn basic
this indicator of growth of virus refers to a process wherein viruses binds to RBC
hemadsorption
Viral envelope proteins may bind glycoproteins expressed on the surface of
erythrocytes
Addition of
hemadsorption
hemadsorption
Addition of __ to the cultures
guinea pig erythrocytes
what virus shows HEMADSORPTION as its indicator of cell culture growth
influenza and parainfluenza viruses
Growth of the first will inhibit the infection of the second virus by
interference
interference
Phenomenon for which a cell infected by a virus becomes resistant
toward a second outcoming infection by a superinfectant virus.
interference
Non-cytopathogenic virus tested with known cytopathogenic virus
interference
refers to a process and an indicator of cell culture growth predominantly practiced by Tumor forming (oncogenic) viruses
transformation
Growth appears in a piled-up fashion
producing microtumors
transformation
Tumor forming (oncogenic) viruses examples
herpes viruses
adenoviruses
hepadnavirus
papovavirues
retroviruses
Technique used to visualize and localize specific antigens (viral
proteins) within cells.
immunofluorescence
Gives positive results earlier than other methods
immunofluorescence
stain used in immunofluorescence
Fluorescein Isothiocyanate (FITC)
2 types of immunofluorescence
direct and indirect
this type of immunofluorescence use A single labeled antibody is used to
directly detect the target viral antigen
direct immunofluorescence
this type of immunofluorescence in which A primary antibody is followed by a
labeled secondary antibody, amplifying
the signal and increasing sensitivity
indirect immunofluorescence
a more rapid but less sensitive type of immunofluorescence
direct IMF
this type of immunofluorescence is best suited to large quantities of virus are suspected or when high quality, concentrated monoclonal antibodies are used
direct IMF
used when lower quantities of virus are
suspected, such as detection of
respiratory viruses in specimens from
adult patient
indirect IMF
Molecular-based assays example
polymerase chain reaction
Provide rapid, sensitive, and specific methods of detection
DETECTION OF VIRUS-SPECIFIC NUCLEIC ACID
Identified by detection of viral enzymes, such as reverse transcriptase in a virus ____ in the culture fluid
retroviruses
Using antibodies to detect viral antigens or antibodies in the culture medium
detection of enzymes
disadv of nucleic acid tsting
expensive
more specialized facilities needed
easily contaminated
low viral load but still have remnants will cause false positive
Powerful technique used to visualize at a very high resolution of virus
electron microscope
used to Study the structure, morphology and size of viral particles in d
electron microscope
passes a beam of electrons through a thin specimen. The electron that pass
through interact with the specimen, creating an image
Transmission Electron Microscopy (TEM)
Useful in studying the internal structure of viruses. It can reveal presence of viral
capsids, nucleic acid and other internal components
Transmission Electron Microscopy (TEM)
2 VIRAL ASSAY
total virus particles
infectious virion assay
2 methods under Total virus particles
electron microscopy
hemagglutination
2 methods under infectious virion assay
quantal assays
quantitative infectivity assay
Step 1 in electron microscopy
Negative Staining-virus suspension is mixed with a negative stain
Step 2 in electron microscopy
: Latex Particle Addition-a known concentration of latex particles of a specific size is
added to the virus suspension. The latex particles serve as a visual reference
Step 3 in electron microscopy
3: Electron Microscopy-The sample is examined under an electron microscope. The virus
particles and latex particles can be distinguished based on their size and
appearance
Step 4 in electron microscopy
Counting-virus particles and latex particles are counted in a specific area of the
electron micrograph. The ratio between the two can be used to estimate the
concentration of the virus particles in the original suspension
A convenient method of quantitation for certain viruses particularly that possess
hemagglutinin proteins
Hemagglutination
Hemagglutination is not a very sensitive indicator of the presence of small amount of
virus particles.
true or false
true
steps in Hemagglutination
Step 1: Virus Dilution-Virus suspension is serially diluted
Step 2: Red blood cell addition-fixed volume of red blood cell is added to each dilution
incubate at 37°C`
Step 3: Observation of hemagglutination-if virus concentration is high enough, red blood
cells will clump together
Step 4: Endpoint Determination-Highest dilution that still produces hemagglutination is
determined
Used to measure the “all-or-none” response of a population of virus
quantal assay
Only indicates presence or absence of infectious viruses
quantal assay
Quantal assays of infectivity can be carried out in __ for those
viruses
animals, eggs or tissue culture
The virus sample is serially diluted to determine the lowest concentration that can still
produce infection
quantal assay
Destruction of the host cell, embryo or animal or the appearance of CPE in cell cultures
quantal assay
The titer of the original virus suspension is expressed as the 50% infectious
dose (ID50) or the 50% lethal dose (LD50) per milliliter
ID50 or LD50:
measure the actual number of infectious
particles in the inoculum
Quantitative assays
quantitative infectivity assay
Two methods are available—
plaque assay in monolayer cell culture
and pock assay on chick embryo CAM
Viral suspension is added to a monolayer of cultured cells in a bottle or petri dish allowing time for
absorption
plaque assay
Each infectious viral particle gives rise to a localized
focus of infected cells that can be seen with the naked
eye. - Known as ‘plaques’ and each plaque indicates
an infectious virus
plaque assay
Viral suspension is added to a monolayer of cultured
cells in a bottle or petri dish allowing time for
absorption,
Removed and replaced with a solid agar gel to
prevent virus spreading throughout the culture
plaque assay
___, form pocks when
inoculated onto the chorioallantoic
membrane of an embryonated egg.
Herpes and vaccinia
in this assay, viruses can be assayed by
counting the number of pocks formed on
cam by appropriate inocula of virus
pock assay
