Virology for fish viruses:Cell cultures according to EU

manuals; accreditation and validation

procedures

Snježana Zrnčić, PhD, DVMTCDC/TCCT Consultant No 2 –

Diagnosticszrncic@irb.hr

According to EU Diagnostic Manual virus isolation on cell culture is golden standard for listed disease diagnosticsSuggested 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)

Fish virus isolation on cell culture 1

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 75Izračun: (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 STANICENpr. broj izbrojenih stanica je 31Izračun: (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. 

Cell lines for virus isolation are propagation in open systems; 24 of 96 wells plate with flat bottomOpen 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 growingIt is compulsory inoculate material to one epithelial (EPC or FHM) incubated on 240C and one fibroblast (BF2 or RTG 2) incubated on 220C cell lineorgan 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 2

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 lineInoculated 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 controlsCell lines used for virus isolation should be tested to susceptibility to VHSV and IHNV each 6 monthsInoculated cell cultures should be checked by invert microscope at least 3 times a week for the presence of CPEIf obvious CPE appeared supernatant should be submitted to identification

Fish virus isolation on cell culture 3

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 4

Inoculated culture are incubated another 7 to 10 daysIf toxic effect appear in first three days, subcultivation must be perform at this stage followed by 7 days incubationIf bacterial contamination appears, supernatants should be centfriguded at 5000 rpm at 2-50C during 20 minutes, succeeded by filtrationIf no CPU appears during second 7 days incubation the sample should be considered NEGATIVEIf evidence of CPU appears in cell lines, supernatants should be submitted to identification by neutralisation, ELISA, IFAT, RT-PCR

Fish virus isolation on cell culture 5

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.

ACCREDITATION

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 non-conformities, 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 1

RequirementsWritten protocols (SOPs, working instruction for sampling,

sample preparation, cell culture preparation, for disposal of tested material and used consumables etc.

Lab equipmentCertified calibration for equipmentCertified calibration of laboratory environmentSecurity measures (measures for avoiding contamination

etc.)Written responsibilities and duties of educated Lab membersChecking of lab stuffSigned records for each step of testingRecord keepingMethod validation

ACCREDIATION OF VIROLOGICAL EXAMINATION OF FISH MATERIAL ON EPC AND BF2 CELL LINE

2

TECHNICAL REQUIREMENTSLaboratory equipmentLAF benchCentrifuge with coolingMicroscopeIncubators 15oC, 22oC,

24oCFreezer -80oCFreezer -20oCRefrigeratorMultichannel 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

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 96-well 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

Validation of method

 

OZNAKA

VIRUSA

I POZNATI

TITAR

 

STANIČNA

LINIJA I

POZNATI

TITAR

 

OČITANI TITAR

Ploča

1

Ploča

2

Ploča

3

Ploča

4

Ploča

5

 

V 01

VHSV –DK-

5151 

 

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

 

V 13

VHSV –1p8 

 

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+

0.3

 

8.6E+

0.3

 

5.9E+

0.3

 

4.0E+

0.3

 

2.7E+

0.3

 

V 03

VHSV –DK-

F1 

 

EPC

(2.7E+0.5)

 

5.9E+

0.5

 

2.7E+

0.6

 

5.9E+

0.5

 

2.7E+

0.6

 

5.9E+

0.5

 

BF-2

(2.7E+0.6)

 

4.0E+

0.5

 

2.7E+

0.6

 

4.0E+

0.5

 

1.9E+

0.6

 

4.0E+

0.5

CPE Pozitivan x/6

Razrjeđenje

-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

Prilog 3. SOP Z-IV-3-R-07: Određivanje titra u 10-strukim razrjeđenjima s ponavljanjem u 6 jažica (inokulum 25 µl)