Virology for fish viruses:
Cell cultures according to EU manuals;
accreditation and validation procedures
Snježana Zrnčić, PhD, DVM
TCDC/TCCT Consultant No 2 – Diagnostics
zrncic@irb.hr
Fish virus isolation on cell culture 1
According to EU Diagnostic Manual virus isolation on cell
culture is golden standard for listed disease diagnostics
Suggested susceptible permanent cell lines are:
 EPC - Epithelioma papulosum cyprini - epithelial (Fijan et al. 1983)
 BF – Bluegill fin – fibroblast (Wolf and Quimby 1962)
 FHM – Fathead minnow – epithelial (Gravell & Mallsberger 1965)
 RTG 2 – Rainbow trout gonads – fibroblast (Wolf and Quimby
1962)
 Each cell line is growing at 20 to 300C in suitable medium
(EMEM – Eagle’s minimal essential medium with Earle’s salt
supplemented with 10% of foetal bovine serum and
antibiotics)
 Cell lines are propagating and maintaining in closed bottles
for cell line cultivation (different producers)
Prilog 3. Z-IV-3-RU-14 POSTUPAK ZA ODREĐIVANJE POTREBNOG BROJA
STANICA PRI PRIPREMI 96-JAŽIČNIH PLOČA SA STANIČNIM KULTURAMA
1 MALA BOCA (25cm2) : 3 ploče s 96-jažica
1 96 jažična ploča : 1/3 stanica iz jedne male boce (25cm2)
1/3 x 2 ml gojilišta = 0,7 ml
(za 1 96 jažičnu ploču potrebno je 15 ml EMEM+Tris u koji se resuspendira 0,7 ml tripsiniziranih s
male boce)
BROJENJE: 1 kap suspenzije se stavi u Burkerovu komoricu, prekrije pokrovnicom i izbroji 16
kvadratića (1 dvostruko obilježeno područje)
BROJ IZBROJENIH STANICA MORA BITI 45!
A) PREGUSTE STANICE
Npr. broj izbrojenih stanica je 75
Izračun:
(75-45) x 100/45 = 66,7%
ZA POVOLJNU GUSTOĆU OD 45 STANICA PO IZBROJENIM KVADRATIĆIMA IZRAČUN JE
SLIJEDEĆI:
15 ml EMEM+Tris x 66,7%/100 = 10,00 ml
U 15 ml EMEM+Trisa s resuspendiranim stanicama potrebno je dodati još 10 ml čistog
EMEM+Trisa.
B) PRERIJETKE STANICE
Npr. broj izbrojenih stanica je 31
Izračun:
(45-31) x 100/45 = 31,1%
ZA POVOLJNU GUSTOĆU OD 45 STANICA PO IZBROJENIM KVADRATIĆIMA IZRAČUN JE
SLIJEDEĆI:
31,1% x 0,7 ml = 0,22 ml
U 15 ml EMEM+Trisa s resuspendiranim stanicama potrebno je dodati još 0,22 ml resuspendiranih
stanica.
Fish virus isolation on cell culture 2
Cell lines for virus isolation are propagation in open systems;
24 of 96 wells plate with flat bottom
Open systems should be buffered with addition of Tris-HCl or
Hepes or no buffer added but incubation should be in
incubator with CO2, pH must be 7.6±0.2
Cell cultures used for inoculation of material for virus isolation
should be young (4 to 48 hours) and actively growing
It is compulsory inoculate material to one epithelial (EPC or
FHM) incubated on 240C and one fibroblast (BF2 or RTG 2)
incubated on 220C cell line
 organ suspension for inoculation of cell lines should be
antibiotic treated of filtered through filter with pore 450 µm
and diluted 1:10 and 1:100 – dilution are prepared on the
plate with round bottom
Fish virus isolation on cell culture 3
For each dilution a minimum of about 2 cm2 cell area
corresponding to a well of 24wells plate or 4 wells of 96 wells
plate should be utilized on each cell line
Inoculated cell lines are incubated at 15±20C for 7 to 10 days;
each plate should have some wells of negative control and
positive VHSV/IHNV controls
Cell lines used for virus isolation should be tested to
susceptibility to VHSV and IHNV each 6 months
Inoculated cell cultures should be checked by invert
microscope at least 3 times a week for the presence of CPE
If obvious CPE appeared supernatant should be submitted to
identification
Fish virus isolation on cell culture 4
 If there is no CPE the primary incubation for 7 to 10 days,
subcultivation is performed to fresh cell cultures utilising a cell
area similar to that of the primary culture
 Aliquots of medium (supernatant) from all cultures/wells
constituting the primary culture are pooled according to cell line 7
to 10 days after inoculation
 The pools are then inoculated into homologous cell cultures
undiluted and diluted 1:10 (resulting in final dilutions of 1:10 and
1:100, respectively, of the supernatant)
 The inoculation may be preceded by preincubation (1 hour at 150C
or 18 hours at 40C) of the dilutions with the antiserum to IPN virus
(equal parts of antisera to different serotyse of IPN)
 The aim of this pretreatment is to shorten the duration of
virological examination
 It is very useful in countries with more than 50% of farm infected
Fish virus isolation on cell culture 5
Inoculated culture are incubated another 7 to 10 days
If toxic effect appear in first three days, subcultivation must
be perform at this stage followed by 7 days incubation
If bacterial contamination appears, supernatants should be
centfriguded at 5000 rpm at 2-50C during 20 minutes,
succeeded by filtration
If no CPU appears during second 7 days incubation the sample
should be considered NEGATIVE
If evidence of CPU appears in cell lines, supernatants should
be submitted to identification by neutralisation, ELISA, IFAT,
RT-PCR
ACCREDITATION
 ISO/IEC 17025 Accreditation Resources
 Accreditation is defined as a procedure by which an authoritative body gives formal
recognition that a laboratory is competent to perform specific tasks. Lab
accreditation recognizes a lab's technical capability and is usually specific for tests of
the systems, products, components, or materials for which the lab claims proficiency.
Accreditation allows a lab to determine whether it is performing its work correctly
and according to appropriate standards. This does not guarantee that a given
analytical result is correct, but it does establish standards that must by met and a
framework approach to detect nonconformities when they occur. Good labs will
have defensible results, but accreditation means that the results are defensible to a
recognized standard that does not change when lab personnel or circumstances
change.
The aims of ISO 17025 are to:
1. Provide a basis for use by accreditation bodies in assessing the competence of
laboratories.
2. Establish general requirements for demonstrating lab compliance to perform
specific tests or calibrations.
3. Assist in the development and implementation of a lab's quality system.
ACCREDIATION OF VIROLOGICAL
EXAMINATION OF FISH MATERIAL ON EPC
AND BF2 CELL LINE 1
Condition that should be ensured are divided into 2 groups:
 Management conditions (organisation of lab, management
organisation, documents management, evaluation of
quotations and contracts, supply by utensiles and services,
customer services, complints, management with nonconformities, improvement, corrective action, preventive
actions, record management , internal audits, evaluation of
the management system
 Technical conditions for analytical labs (stuff, accomodation
and environmental conditions, analytical methods,
calibration and validation, equipment, traceability, sampling,
manipulation with samples, warranty for quality of testing
and calibration, reporting
ACCREDIATION OF VIROLOGICAL EXAMINATION OF
FISH MATERIAL ON EPC AND BF2 CELL LINE 2
Requirements
Written protocols (SOPs, working instruction for sampling,
sample preparation, cell culture preparation, for disposal of
tested material and used consumables etc.
Lab equipment
Certified calibration for equipment
Certified calibration of laboratory environment
Security measures (measures for avoiding contamination etc.)
Written responsibilities and duties of educated Lab members
Checking of lab stuff
Signed records for each step of testing
Record keeping
Method validation
TECHNICAL REQUIREMENTS
Laboratory equipment
LAF bench
Centrifuge with cooling
Microscope
Incubators 15oC, 22oC, 24oC
Freezer -80oC
Freezer -20oC
Refrigerator
Multichannel pipettes
Laboratory supplies and
consumables
 Polistyren sterile pipettes 1,2
5,10ml and tubes
 24, 96 wells plates with flat
bottom
 Cell culture plastic bottles
 Sterile tips for pipettes 100, 1000 μl
 EMEM
 FBS
 Trypsin
 Rubber gloves
 HEPES
 Disinfectants
Validation of method
1. Mycoplasma testing – twice a year or more if there is any
doubt
2. Testing of cell lines sensitivity for viruses
 Scope – sensitivity of EPC and BF2 cell lines for listed viruses
(VHSV – at least two types, one pathogenic for freshwater
fish and marine isolate and IHNV)
 Procedure – for each cell line and each virus 15 half of 96well plate should be prepared, 10-fold dilution of virus with
known titer, incubation at 15oC for 7 days
 determining the titer, data evalution and conclusion on
sensitivity; if there is a decrease of sensitivity, a new batch
of cell line is needed either from liquid nitrogen, or ask from
CRL or some other reliable lab
OZNAKA
STANIČNA
VIRUSA
LINIJA I
Ploča
Ploča
Ploča
Ploča
Ploča
1
2
3
4
5
EPC (1.9E+0.3)
2.7E+0.3
4E+0.3
1.9E+0.3
4E+0.3
2.7E+0.3
BF-2 (2.7E+0.3)
1.9E+0.3 2.7E+0.3 1.9E+0.3 1.9E+0.3 2.7E+0.3
EPC (2.7E+0.2)
1.3E+0.4 2.7E+0.4 1.9E+0.4 1.3E+0.4 8.6E+0.3
BF-2 (1.9E+0.4)
4.0E+0.
I POZNATI TITAR POZNATI TITAR
V 01
VHSV –DK-5151
V 13
VHSV –1p8
OČITANI TITAR
3
V 03
VHSV –DK-F1
EPC (2.7E+0.5)
8.6E+0. 5.9E+0.3 4.0E+0. 2.7E+0.3
3
3
5.9E+0.5 2.7E+0.6 5.9E+0.5 2.7E+0.6 5.9E+0.5
Prilog 3. SOP Z-IV-3-R-07: Određivanje titra u 10-strukim razrjeđenjima s
ponavljanjem u 6 jažica (inokulum 25 µl)
Razrjeđenje
CPE
Pozitivan
x/6
-1
-2
-3
-4
-5
-6
-7
-8
-9
-10
1
1.9x102
1.9x103
1.9x104
1.9x105
1.9x106
1.9x107
1.9x108
1.9x109
1.9x1010
1.9x1011
2
2.7x102
2.7x103
2.7x104
2.7x105
2.7x106
2.7x107
2.7x108
2.7x109
2.7x1010
2.7x1011
3
4x102
4x103
4x104
4x105
4x106
4x107
4x108
4x109
4x1010
4x1011
4
5.9x102
5.9x103
5.9x104
5.9x105
5.9x106
5.9x107
5.9x108
5.9x109
5.9x1010
5.9x1011
5
8.6x102
8.6x103
8.6x104
8.6x105
8.6x106
8.6x107
8.6x108
8.6x109
8.6x1010
8.6x1011
6
1.3x103
1.3x104
1.3x105
1.3x106
1.3x107
1.3x108
1.3x109
1.3x1010
1.3x1011
1.3x1012