SEMR411: SEMINAR 1 LECTURE

14.1 Cell culture

A.Y. 2021-2022 | Second Semester | Finals
I. VIRUSES o These cells are examined for cytopathogenic effect
• Submicroscopic, obligate intracellular parasites among (CPE)
the smallest of all infectious agents. • The supernatant is used as a representative of possible
• Very specific and each has a limited number of hosts it virus in the sample.
can infect (viral tropism) o Inhibitory substances are added to get rid of
• May be classified by type of disease but are most possible contaminants.
commonly categorized based on the type of nucleic acid.
• Viruses that causes human disease II. CELL CULTURES
o There are hundreds of viruses that cause disease in • Viruses require living cells, suitable host cells, culture
human which ranges in its severity from being media and techniques in cell culture maintenance in
asymptomatic to fatal. order for it survive and replicate outside its host.
o Viruses of human medical importance comprise 4 • Host cells originate as few cells and grow in a single
orders, 25 families, 13 subfamilies and 66 genera. monolayer on the sides of the glass or plastic tubes.
o Alternative: Multiwell microtiter plates.
A. LABORATORY DIAGNOSIS • The cells are kept moist and are supplied with nutrients
• Clinical virology laboratory services have grown at a rapid by keeping them continuously immersed in a cell culture
state which was because of the introduction of: medium.
o Virus-specific antiviral drugs • Cell culture tubes can be incubated in a stationary rack
o The commercial availability of reagents or roller drum.
• The development of rapid diagnostic techniques using • Once inoculated, cell cultures are incubated for 1-4
conventional methods such as: weeks, depending on the suspected viral agents.
1. Fluorescence microscopy and enzyme • Periodically the cells are inspected microscopically with
immunoassays an inverted light microscope for CPE.
2. The ready availability of cell lines for virus detection • Virus-induced CPE presents two other important
in cell cultures considerations:
3. The widespread use of nucleic acid amplification o Rate at which the CPE progresses
tests for detecting viral infections. o If the type of cell culture in which the virus grows
may be used for presumptive identification.
B. SPECIMEN SELECTION AND COLLECTION • An example of CPE rate progression is observed in
SPECIMEN SELECTION herpes viruses:
o HSV: CPE progresses rapidly
• Depends on the specific disease syndrome, viral agents
o VZV and CMV: progresses slowly.
suspected and time of the year.
• Cell culture may serve as an indicator of presumptive
• Based on disease is difficult as some diseases may
identification of virus can be observed in poliovirus and
produce symptoms distant from the primary inoculation
echovirus
site.
o Rhesus monkey kidney (RMK) cells: produce
• Based on the suspected viral agent is complicated due
similar CPE
to a clinical syndrome may be caused by many different
o Continuous cell lines: no induced CPE for
viruses.
echovirus.
SPECIMEN COLLECTION A. CLASSIFICATION OF CELL CULTURES
• Specimen for virus detection should be collected as early • Cell cultures are classified into three types based on:
as possible after the onset of symptomatic disease. o Origin
• However, other factors must be taken in account such as o Chromosomal characters
PATA the patient’s immune status, age, the type of virus and o Number of generations through which they can be
amount of systemic involvement. maintained.
• The selection of the type of cell culture depends on the
C. VIRUS DETECTION METHODS particular virus to be isolated
• The following are different methods for the detection of A cell culture becomes a cell line once it has been passed or
viruses: subcultures, in vitro.
o Cytology and Histology
o Electron microscopy B. MEDIA USED FOR CELL CULTURE
o Immunodiagnosis (Antigen Detection) GROWTH MEDIUM MAINTENANCE MEDIUM
o Enzyme-Linked Virus-Inducible System • Serum-rich nutrient • Contains less serum
o Molecular Methods medium (10% fetal, (0-2%)
o Cell Culture newborn or • Used to keep cells in a
agammaglobulinemic steady state of
D. VIRUS ISOLATION IN CELL OR TISSUE calf serum) designed to metabolism
CULTURE support rapid cell • Fetal, newborn, or
• Virus isolation in culture remains gold standard for growth agammaglobulinemic
the isolation of many viruses. • Used to initiate the calf serum is used to
• Cell cultures are animal or human cells grown in vitro growth of cells when avoid inhibitors of virus
that have lost their differentiation cell cultures are replication and it is free
prepared in-house or to of mycoplasmas

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 14.1 Cell culture
feed purchased cell 3. Hemadsorption
cultures 4. Interference
• Both media are prepared with Eagle’s minimum 5. Transformation
essential medium (EMEM) in Hank's or Eagle’s 6. Immunofluorescence
balanced salt solution (HBSS or EBSS) 7. Detection of Virus-specific Nucleic Acid
o HBSS: carbon dioxide 8. Detection of Enzymes
o EBSS: ambient air 9. Electron Microscopy
• Antimicrobials are also added to prevent bacterial
contamination A. CYTOPATHOGENIC EFFECT (CPE)
o Vancomycin (10 mg/mL) • Refers to cellular damage or changes in the cellular
o Gentamicin (20 mg/mL) structure.
o Amphotericin (2.5 mg/mL) • These changes can be readily observed by microscopic
examination of the cultures.
C. CELL CULTURE FOR ISOLATION OF VIRUSES • These changes are known as ‘cytopathic effects’ (CPE)
• Inoculated cell cultures should be incubated immediately and the viruses causing CPE are called ‘cytopathogenic
at 35°C viruses’
• After allowing the virus to adsorb to the cell monolayer for
12 to 24 hours, the remaining inoculum and culture MAIN TYPES OF CPE
medium commonly are removed and replaced with fresh Rounding of Viral replication may lead to nuclear
maintenance medium. cells pyknosis, rounding, refractility, and
o Maintenance medium should be changed degeneration
periodically to provide fresh nutrients to the cell Cell necrosis Enteroviruses produce rapid CPE with
• Incubation should be continued for 5 to 28 days, and lysis crenation of cells and degeneration of the
depending on the suspected agent entire cell sheet
• Blind passage is the passing of cells and fluid to a Syncytium Infected cells fuse with neighboring
second cell culture tube formation infected or uninfected cells to form giant
cells containing several nuclei
SHELL VIAL CENTRIFUGATION-ENHANCED Discrete focal Observed in Herpes virus
(SVCE) CULTURES degeneration
• Rapid modification of conventional cell culture Rounding Produces large granular clumps
• Virus is detected more quickly and resembling bunches of grapes
• CPE can be identified within 1 to 2 days by detecting early aggregation
produced viral antigens.
• Can be used to detect most viruses that grow in QUANTITATION OF CELL CULTURE CYTOPATHIC
conventional cell culture. EFFECTS
o Best for viruses requiring relatively long incubation QUANTITATION INTERPRETATION
period before producing CPE. Negative Uninfected monolayer
• Advantage: speed Equivocal (±) Atypical alteration of monolayer
• Disadvantage: only a single type of virus can be detected involving a few cells
per shell vial. 1+ 1% - 25% of monolayer exhibits
cytopathic effects (CPE)
D. DETECTION OF VIRUS GROWTH IN CELL 2+ 25% - 50% of monolayer exhibits CPE
CULTURE 3+ 50% - 75% of monolayer exhibits CPE
• Viruses are most often detected in cell culture by the 4+ 76% - 100% of monolayer exhibits
recognition of CPE CPE
o Cells infected by virus have morphological changes
and lyse or detach from the glass surface while dying B. METABOLIC INHIBITION
o Viruses have distinct CPE • When viruses grow in cell cultures, cell metabolism is
• Preliminary/ presumptive identification of a virus can be inhibited and there is no acid production.
made based on: • This can be made out by the color of the indicator (phenol
o Cell line that supports viral replication, red) incorporated in the medium
o How quickly the virus produced CPE
o Description of the CPE C. HEMADSORPTION
• Confirmatory tests or definitive identification may be done • Guinea pig RBCs are added to the culture where RBCs
by performing additional testing such as: will adsorb onto the surface of the cells if the viruses are
o Fluorescent-labeled antisera multiplying
o Acid lability • This reaction becomes positive before CPE are visible
• Some viruses produce little to no CPE it can be detected • Non-CPE inducing viruses can be detected by this
through hemadsorption method
o Performed by adding guinea pig RBCs to the cell
culture tube, followed by washing. D. INTERFERENCE
o A ring of RBCs around infected cell will be observed • The growth of a non-cytopathogenic virus in cell culture
can be tested by the subsequent challenge with a known
III. METHODS FOR VIRUS DETECTION IN CELL cytopathogenic virus.
CULTURE • The growth of the first will inhibit infection by the second
1. Cytopathic Effect virus by interference
2. Metabolic Inhibition
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 14.1 Cell culture

E. TRANSFORMATION
• Tumor forming (oncogenic) viruses induce cell
‘transformation’ and loss of contact inhibition, so that
growth appears in a piled-up fashion producing
microtumors

F. IMMUNOFLUORESCENCE
• Cells from virus infected cultures can be stained by
fluorescent conjugated antiserum and examined under
the UV microscope for the presence of virus antigen

G. DETECTION OF VIRUS-SPECIFIC NUCLEIC

ACID PCR
• Molecular-based assays, such as polymerase chain
reaction, provide rapid, sensitive, and specific methods of
detection.

H. DETECTION OF ENZYMES
• The virus isolate can be identified by detection of viral
enzymes, such as reverse transcriptase in retroviruses, reverse transcriptase (retrovirus)
in the culture fluid

I. ELECTRON MICROSCOPY
• Viruses have distinctive appearances and can be
detected by electron microscopy of ultra-thin sections of
infected cells

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14.1 Cell culture SEMR411 LEC

IV. ISOLATION AND IDENTIFICATION OF COMMONLY CLINICALLY ENCOUNTERED VIRUSES

SIGNS/SYMPTOMS SPECIMEN CULTURE CPE DESCRIPTION RATE OF IDENTIFICATION
GROWTH AND COMMENTS
ADENOVIRUS • Affects the respiratory tract, • Throat swabs, • Can be inoculated Rounding and 2- 10 days • Confirm by FA
eye, and gastrointestinal (GI) nasal washings, using cell culture with aggregation of infected test, IFA or EIA
tract, with lesser involvement conjunctival swabs various epithelial cell cells in grapelike • Serotype by
of the urinary tract, heart, or scrapings, or lines; clusters cell culture
central nervous system feces o A-549, neutralization
(CNS), liver, pancreas, and o HEp-2,
genital tract. o He-La cells.
o HEK cells
CYTOMEGALOVIRUS • Most CMV infections are • Urine, saliva, tears, • Can be isolated Discrete, small foci of 5-28 days • Distinct CPE
(CMV) asymptomatic in the milk, and semen, using cell culture rounded cells sufficient to
immunocompetent host but and vaginal with: identify
can manifest themselves as a secretions o Human diploid • Confirm by FA
self-limiting, infectious fibroblasts (human test
mononucleosis-like illness, embryonic lung or
with fever and hepatitis. human foreskin
fibroblasts)
o Shell vial cultures:
(1 day)
ENTEROVIRUS • Various conditions, including • Throat swabs, • No single cell line Characteristic refractile 2-8 days • Confirm by FA
fever of unknown origin, feces, CSF, urine, supports the growth angular or tear-shaped test Stable at
aseptic meningitis, paralysis, blood, and of all types of CPE; progresses to pH 3
sepsis like illness, conjunctival swabs, enteroviruses. involve entire
myopericarditis, pleurodynia, depending on the • A variety of primate monolayer
conjunctivitis, exanthemas, site of the infection. and human cell lines
pharyngitis, and pneumonia may be used for virus
isolation
HERPES SIMPLEX • HSV-1 causes oral herpes, • Can be isolated • Aspirates from Rounded, swollen 1-3 (may • Distinct CPE
VIRUS (HSV) gingivostomatitis, ulcerative using cell culture or vesicles, open refractile cells; take up to sufficient to
mouth lesions, and fever shell vial (SVCE) lesions, or host cells occasional syncytia, 7) identify
blisters. with: collected from especially with HSV-2; • Confirm by FA
• HSV-2 produces 80% to 90% o HeLa cells infected sites. rapidly involves entire test
of all cases of genital herpes, o Human monolayer
a common sexually embryonic
transmitted disease (STD). fibroblasts
• Neonatal HSV-2 infections o Hep2 A549
may result if the infant o Rabbit kidney
acquires infection during cells
delivery from an actively
infected mother.

INFLUENZA • Fever • Optimal specimens • Grow in amniotic Destructive 2-10 days • Detect by
• nonproductive cough are collected from cavity of degeneration with hemadsorption
• sore throat the posterior embryonated swollen, vacuolated or
• rhinitis nasopharynx. chicken eggs and cells hemagglutinati
• headache various mammalian
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14.1 Cell culture SEMR411 LEC

• malaise and • Other respiratory cell culture lines, on with guinea

• myalgia samples including such as: pig RBCs
nasal aspirates, o PMK • Identify by FA
nasal wash, throat o MDCK Cells test
swabs, and throat • Rapid culture assays
washes. can be performed
using:
o If staining of
infected
monolayers grown
in shell vials
o flat-bottomed wells
of microtiter plates.
MUMPS • Causes parotitis, • Saliva Can be inoculated using CPE usually absent; 5- 10 days • Detect by
accompanied by a high • Urine cell culture with: syncytia occasionally hemadsorption
temperature (fever) and • CSF, and o Vero or LLC-MK2 seen with guinea pig
fatigue • pharyngeal cell: To be more RBCs
secretions successful than • Confirm by FA
those with HEp2 or test
HeLa cell lines
PARAINFLUENZA • Parainfluenza 1 is the most Mucous membranes of Can be inoculated using CPE usually minimal or 4- 10 days • Detected by
common cause of croup, and the respiratory tract cell cultures with: absent hemadsorption
• Parainfluenza 3 is second in (nasopharyngeal • PMK cells with guinea pig
prevalence to RSV in washing) • LLC-MK2 cells • RBCs Identify
respiratory diseases such as by FA test
bronchiolitis and pneumonia
RESPIRATORY • Causes croup, bronchitis • Nasal aspirates Grows readily in Syncytia in HEp-2 cells 3 - 10 days • Distinct CPE in
SYNCYTIAL VIRUS • bronchiolitis, and • throat swabs continuous epithelial cell HEp-2 cells
(RSV) • interstitial pneumonia • nasopharyngeal lines, such as HEp2 sufficient for
specimens, or presumptive
• sputum. identification
CONTINOUS EPIT • Confirm by FA
test
RHINOVIRUS • Most frequent cause of the • Throat swabs, Can be inoculated using Characteristic refractile 4- 10 days • Labile at pH 3
common cold. Self-limiting, nasal washings, cell cultures of human rounding of cells; in • Growth optimal
with symptoms of a mild conjunctival swabs, origin such as: PMK, CPE is identical to at 32°C to
respiratory illness, including scrapings, or feces • MRC5 that produced by 33°C.
nasal congestion. • WI38 enteroviruses
VARICELLA ZOSTER • Chicken pox (varicella); Scrapings of fresh • Can be inoculated Discrete foci of 5 - 28 days Confirm by FA test.
VIRUS (VZV) shingles (zoster).; mild febrile vesicular lesion using cell cultures rounded, swollen,
illness, rash, and vesicular with fibroid cells such refractile cells; slowly
lesions. as: involves entire
o MRC-5, monolayer
o HF
o A-549
• Shell vial cultures
may also be used
with coverslips with
MRC-5 cell
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