Virology for fish viruses: Cell cultures according to EU manuals; accreditation and validation procedures Snježana Zrnčić, PhD, DVM TCDC/TCCT Consultant No 2 – Diagnostics

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 30 0 C 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 (25cm 2 ) : 3 ploče s 96-jažica 1 96 jažična ploča : 1/3 stanica iz jedne male boce (25cm 2 ) 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.

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 CO 2, 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 24 0 C and one fibroblast (BF2 or RTG 2) incubated on 22 0 C 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 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 line Inoculated cell lines are incubated at 15±2 0 C 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 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 15 0 C or 18 hours at 4 0 C) 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 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 C 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 Fish virus isolation on cell culture 5

 ISO/IEC 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 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

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 ACCREDIATION OF VIROLOGICAL EXAMINATION OF FISH MATERIAL ON EPC AND BF2 CELL LINE 2

TECHNICAL REQUIREMENTS Laboratory equipment  LAF bench  Centrifuge with cooling  Microscope  Incubators 15 o C, 22 o C, 24 o C  Freezer -80 o C  Freezer -20 o C  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

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 E+0.3 4E E+0.3 BF-2 (2.7E+0.3) 1.9E E E E E+0.3 V 13 VHSV –1p8 EPC (2.7E+0.2) 1.3E E E E E+0.3 BF-2 (1.9E+0.4) 4.0E E E E E+0.3 V 03 VHSV –DK-F1 EPC (2.7E+0.5) 5.9E E E E E+0.5 BF-2 (2.7E+0.6) 4.0E E E E E+0.5

Prilog 3. SOP Z-IV-3-R-07: Odre đ ivanje titra u 10-strukim razrje đ enjima s ponavljanjem u 6 jažica (inokulum 25 µl)