SOP: Virus Isolation
Date Issued: Jan 15, 2014
Global Pathogen Laboratory
Emerging pathogen Institute
University of Florida
SOP: Virus Isolation
One Health Center of Excellence
Emerging pathogen Institute
University of Florida
I.
PURPOSE:
This procedure provides details and background for isolation of respiratory viruses,
particularly dengue, chikungunya, West Nile virus and Japanese encephalitis using Vero
cell line.
Consult your supervisor before varying any part of this procedure.
II.
PRECAUTION:
A. Biological issues:
Clinical samples are a biological hazard therefore appropriate precautions must be taken
at all times including working under biosafety cabinet and using all proper personal
protective equipment and.
Disinfect all surfaces and working areas with 10% freshly prepared bleach followed by
70% freshly prepared ethanol. You may use SaniWipes® to wipe internal hood surface
before and after using.
Always autoclave all used material after your work in the day is complete
B. Chemical safety:
Amophotericin B(Fungizone) is mutagenic. Use caution when preparing and using media
that contains this reagent. Always wear gloves and dispose of spent media in the
biohazard waste to be autoclaved and burnt.
C. Required training:
All laboratory personnel working with direct exposure to blood samples need to
complete blood-borne pathogen training.
D. Assay Precaution:
Tissue culture media/cells are easily contaminated with bacteria and fungi.
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Use sterile technique at all times.
SOP: Virus Isolation
Date Issued: Jan 15, 2014
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Global Pathogen Laboratory
Emerging pathogen Institute
University of Florida
Wash your hands and arms with soap before touching tissue culture flasks and
reagents.
Turn on the UV light in the hood 10 minutes prior to working in the hood and 10
min after working with infectious material.
 Wipe anything you want to enter inside the hood with 70% ethanol.
Cell cultures are easily contaminated with viruses that have been passed in cell culture.
To avoid cross contamination:
 Always work with samples of least suspected number of viruses first (i.e. clinical
samples).
 If you open a cryovial or a culture flask with propagated virus there is a high
chance that you have contaminated the hood with viral particles, therefore clean
the surface and open the UV for 10 minutes after working with propagated virus
before working with clinical sample.
 Always use a pipet to transfer material into tubes and run the liquid on the side
of the tube, avoid forming bubbles as you go. Pouring out of flask into tube can
form aerosols, which can contaminate the hood.
 Viruses are highly labile; therefore you must always keep your samples on ice.
 Always use polypropylene tubes instead of polystyrene.
III.
BACKGROUND:
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Dengue virus is a single stranded positive sensed RNA virus belonging to family
Flaviviridae. There are four different serotypes of dengue currently known to infect
humans, DENI-IV. The virus is isolated on VERO cell line causing a cytopathic effect
(CPE) in 2-3 days. The virus is transmitted by Aedes aegypti and Aedes albopictus
mosquitoes.
Chikungunya (CHIKV) virus is a single-stranded positive sensed RNA virus belonging
to family Alphaviridae, with three different genotypes. However, there is currently
only one identified serotype. The virus is also propagates in VERO cells causing CPE
3-4 days post inoculation. In some cases there can be a concurrent infection with
Dengue and CHIKV within the same outbreak and population.
Japanese Encephalitis (JE) is a single stranded positive sensed RNA virus of family
Flaviviridae. JE is classified into four distinct genotypes. The virus is mainly
transmitted by Culex tritaeniorhynchus; however, other culicine mosquitoes can
serve as vectors. There is only one identified serotype.
Global Pathogen Laboratory
Emerging pathogen Institute
University of Florida
SOP: Virus Isolation
Date Issued: Jan 15, 2014
 West Nile virus is a single stranded positive sensed RNA virus of family Flaviviridae,
transmitted mainly by culicine sp. The virus cross-reacts serologically with JE and
Dengue, there are two distinct genotypes with one serotype.
Virus
family
Flavivirus
JEV
Flavivirus
WNV
DENGV
Flavivirus
CHIKV
Alphavirus
IV.
Vector
Hosts
Cell_Line
Vaccine
availability
Culicinae
family
horses, humans,
pigs and
occassionally
birds
Vero, C636, BHK-21,
SPF Chicken embryos
Available
Culicinae
family
Horses, birds and
humans
Vero, C636,SPF chicken
embryos, RK-13
only for
horses
Aedes sp.
Humans
Vero, C636, BHK-21,
embryos
N/A
Aedes sp.
Humans, primates
Vero, C636, BHK-21
N/A
PROCEDURES:
A. General issues:
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Cells should be at least 80% confluent with no sign of contamination or toxicity.
Prepare all worksheets for the clinical samples prior to work.
If you are using tubes for isolation, always consider the last 1 cm in the tube to
assess confluency.
Keep your samples always on ice, viruses are very heat labile.
Vortex your samples gently.
If samples were frozen prior to inoculation, thaw at 37⁰C in a clean water bath
for 2-3 minutes. Once the ice melts immediately transfer samples to a bucket
containing ice.
At least Pre-warm media to room temperature, preferably in a 37⁰C water bath.
Avoid adding cold media to cells.
Clinical samples should always be maintained in virus transport medium (VTM)
B. Infecting cells:
1. Pour off growth media into a discard container.
2. Rinse with PBS to remove traces of FBS.
3. Inoculate 100µL of sample on cells in tubes.
a. If Serum of CSF then sample directly
b. If mosquito or tissue then treat with antibiotic and fungizone
Date Issued: Jan 15, 2014
SOP: Virus Isolation
Global Pathogen Laboratory
Emerging pathogen Institute
University of Florida
4. A thin film of fluid should cover the entire monolayer; cells should not be
allowed to dry. For T-25, T-75 or T-150 inoculate 200, 300 and 500 µL of the
sample, respectively.
5. Rock the flask gently to allow the liquid to fully cover the entire monolayer.
6. Make 3 negative control tubes along with your set. The first should be inoculated
with media, the second should be inoculated only with distilled water, and the
third should remain as it is from the culture lab. All three controls should be used
to assess cell viability and media cleanliness.
7. Incubate at 36⁰C for an hour, in a 5% CO2 incubator.
8. After the hour, inoculate DMEM maintenance media. Volume should be adjusted
according to flask size.
9. Mark the flasks with proper label and place in 36⁰C with 5% CO2 for 7 days.
10. Monitor CPE daily.
11. If contamination is recorded in any of the tubes, discard immediately and mark
in the record book the tube number and the day of contamination.
12. When CPE is observed allow the tube to develop into 2+ - 3+ (50-75%) of
infection. If CPE reaches 100%, virus titer will decrease and viability will decline
rapidly.
C. Harvesting infected cells:
1. Once the cells exhibit approximately 75% CPE, remove from the incubator and
collect all media into a conical tube.
2. Transfer cells with media into a conical tube and centrifuge at 200 X g for 5 min
in a 4⁰C bench top centrifuge.
3. Collect supernatant media and pass through a 0.2 nm sterile filter.
4. Aliquot 100-500 µL of filtered media into a well labeled cryovials.
5. Collect the cell pellet into two cryovials with well-defined labels.
6. Store all tubes at -70⁰C
7. Labels for all tubes should contain the following [cell line – date harvestedpassage history – initials of harvesting technician].
V.
MATERIALS:
A. Virus Transport Medium (broth)
1. To a 500 mL bottle add the following:
a. 10 gm veal infusion broth
b. 2.0 gm of bovine serum albumin (BSA)
c. 0.8 mL gentamicine sulfate
d. 3.2 mL of fungizone
(2.5% final conc.)
(0.5% final conc.)
(100µg/mL final conc.)
(2 µg/mL final conc.)
SOP: Virus Isolation
Date Issued: Jan 15, 2014
e.
2.
3.
4.
Global Pathogen Laboratory
Emerging pathogen Institute
University of Florida
Easy Pure water to 400 mL
Swirl gently to dissolve or let stand at 4⁰C for 1 hour.
Sterile filter.
Label as “virus transport medium” with your name, date prepared, and an
expiration date of 3 months from the date prepared. Store at 4⁰C.
B. Virus Maintenance Media (DMEM):
1. to a 500 mL of DMEM add the following
a. 10 mL of fetal calf serum (FCS) (2% final concentration)
b. 5 mL of Penicillin Streptomycin P/S (1X final concentration)
2. Mark the bottle with today’s date and write on it complete maintenance media
for Vero cell line.
3. Store the bottle away from light in 40C for no longer than 1 month.
C. Equipment:
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36⁰C incubator with 5% CO2.
Biosafety Cabinet type II
Pipette aid
Pipetmen (100-200-1000µL)
-80⁰C Freezer
Vortex mixer
Refrigerator
Cooling centrifuge
Ice bucket
Ice maker
D. Supplies:
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200 and 1000 µL sterile, filter pipet tips.
1, 5, and 10 mL sterile serological pipets
T-25 tissue culture flasks
Tissue culture tubes.
2 mL cryovials (sterile; outside thread)
Freezer boxes
1.5 mL Eppendorf tubes
Kim wipes
3-5 mL syringe
0.2 nm syringe filter, low protein binding, sterile.
 Cryolabels.
Date Issued: Jan 15, 2014
Global Pathogen Laboratory
Emerging pathogen Institute
University of Florida
SOP: Virus Isolation
Virus Isolation Worksheet
15 Jan 2014
Revised:
SAMPLES:
PROTOCOL#:
DATE:
____________________________________________
PURPOSE:
PERFORMED BY: ____________
1. Reagent and Supplies:
Date prepared
1.1. Maintenance media
a.
DMEM media
b.
DPBS
c.
Cell line used
_________
_________
_________
1.2 Cell confluency: _______________
2. Results:
2.1. Summary sheet:
a.
Samples tested
________________________________________________________
b.
Sample Type:
c.
Sample treatment: _______________________________________________________
3. Comments:
________________________________________________________
Global Pathogen Laboratory
Emerging pathogen Institute
University of Florida
SOP: Virus Isolation
Date Issued: Jan 15, 2014
VIRUS ISOLATION – DAILY WORKSHEET
CELL LINE: ___________
Sample Type:________________
Page 1 of __
DATE INOCULATED: ___________ PERSONNEL: ___________ CELL PASSAGE: _________
Tube
Accession
Number
DATE CELLS PASSED: __
Days Post Inoculation
1
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9
Comments
10 11 12
1
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CC
CC
CCC
 = normal; + = 25% CPE; ++ = 50% CPE; +++ = 75% CPE; ++++ = 100% CPE; F = frozen; S = spot slide