Establishment and characterization of fantail goldfish fin (FtGF) cell line from goldfish, Carassius auratus for in vitro propagation of Cyprinid herpes virus-2 (CyHV-2)

Generation of the primary cell culture

A healthy fantail goldfish weighing 20 g was purchased from a local commercial aquarium shop and kept in properly aerated and filtered fish tank. The fish was fed daily and water changes were performed on alternate days. The goldfish was anesthetized using 3- aminobenzoic acid ethyl ester methanesulphonate (MS-222, Sigma-Aldrich, St. Louis, MO, USA) at the dose rate of 150 mg/L of water and the caudal fin was excised after wiping with 70% ethanol. The excised tissue was washed three times in Phosphate Buffer Saline (PBS) (Life Technologies, Carlsbad, CA, USA; Lot number—1967526; Catalogue number—14190-144 500 mL) containing antibiotic–antimycotic solution (200 IU/mL penicillin, 200 μg/mL streptomycin and 0·5 μg/mL amphotericin B) (Life Technologies, Carlsbad, CA, USA; Lot number—2112695; Catalogue number—15240-062 100 mL). The fin tissue was mechanically cut with a fine scalpel into smaller pieces and then seeded in a 25 cm² flask. The PBS was removed and attachment of the tissues to the surface was facilitated by adding 200 µl of fetal bovine serum (FBS) (Life Technologies, Carlsbad, CA, USA; Lot number—42G829K; Catalogue number – 10270-106 500 mL). After 2 h, about 7 mL of Leibovitz’s L-15 (L-15) medium (Life Technologies, Carlsbad, CA; Lot number—2085192; Catalogue number—11415-064 500 mL) containing 20% FBS and 1× concentration of antibiotic antimycotic solution was added to the flask, which was then incubated at 28 °C. The flask was observed daily under an inverted light microscope (Nikon Corporation, Tokyo, Japan).

Subculture and maintenance

After formation of monolayer, the cells were washed with PBS followed by trypsinization with 0.25% trypsin EDTA solution (Life Technologies, Carlsbad, CA, USA; Lot number—2053183; Catalogue number—15400-054 100 mL). The cells were observed under an inverted light microscope. The trypsin EDTA solution was discarded immediately on observing cell detachment. The flasks were gently tapped to release the cells from the surface and L-15 medium with 20% FBS was added to stop the action of trypsin. The cells were subcultured at a split ratio of 1:2. The flasks were observed regularly and the cells were subcultured after attaining 80–90% confluence. After 10 subcultures, the concentration of FBS in medium was reduced to 10%.

Cell growth characteristics

Cell growth studies were conducted at different temperatures and FBS concentrations in L-15 medium. The growth rate assays were conducted at temperatures ranging from 20 to 37 °C and FBS concentrations from 5 to 20%. A monolayer flask at 20th passage was trypsinized and the cells were seeded into different six well plates. The plates were incubated overnight at 28 °C. Thereafter, the plates were incubated at different temperatures of 20, 24, 28, 35 and 37 °C and the growth rate was observed. Cells were harvested from the duplicate wells daily and counted with a Neubauer hemocytometer. A similar study was conducted using different six well plates incubated at 28 °C having different FBS concentrations of 5, 7.5, 10, 15 and 20%. The cells at 25th passage at the concentration of 1 × 10⁵ cells per ml were seeded into 24-well plates and cultured for 7 d at 28 °C. The cells were harvested, the number of cells in each well counted with a hemocytometer, and the average value of three wells at each time was used to plot the growth curve. The growth curve experiment of FtGF cells was repeated two times. Cell growth curves were plotted, and the population doubling time (PDT) was calculated based on the growth curve (Weingartl et al., 2002). Growth characteristic experiment assays were carried out for 8 d, and cell number was represented as mean± standard deviation (SD).

Chromosome analysis

The chromosomal counts for FtGF were undertaken from the 30th, 40th and 56th passage flasks. A flask with 80% confluence was treated with one μg/mL colchicine (Sigma, St Louis, MO, USA) for 4 h at 28 °C and then the supernatant was removed, the cells harvested and resuspended in a hypotonic 0.5% KCl solution for 10 min, and then fixed in a 3:1 methanol : acetic acid solution. The cells were pelleted at 367 g at 4 °C for 10 min and washed four times with methanol and acetic acid solution. Smears were prepared following a conventional drop-splash technique (Freshney, 2010), the slides were stained with 10% Giemsa solution and air dried for 30 min. A total of 100 chromosome spreads were observed and counted under a light microscope (Leica, Germany).

Cryopreservation

The monolayers with 80–90% confluency were used for cryopreservation at various intervals of subculturing (7th, 10th, 20th, 30th and 40th). The media was changed one day prior to cryopreservation. Next day, the cells were trypsinised in PBS and centrifuged at 500 g at 4 °C. at a density of 5 × 10⁶ cells/mL, Briefly, the flask was examined using an inverted microscope to monitor the confluence and to check for microbial contamination if any. The spent cell culture medium was removed. The cell monolayer was washed with a balanced PBS salt solution without calcium and magnesium three times and ensuring the adherence of the cells. One ml of 0.25% trypsin EDTA was added to the 25 cm² surface area flask to cover the monolayer. Excess solution was discarded and the flask was incubated at 37 °C for 4 min. with gentle rocking. The flask was examined for the detachment of cells, the remaining cells were released with gentle tapping and cells were re-suspended in L-15 medium with 20% FBS to deactivate the trypsin. The harvested cells were dispersed by repeated mild pipetting and cells were counted (Freshney, 2010). The cells were suspended in recovery cell-culture freezing medium (Life Technologies, Carlsbad, CA, USA; Lot number—492531; Catalogue number—12648-010 50 mL) aliquoted into two mL sterile cryovials (Nunc), kept at 4 °C for 2 h, −20 °C for 1 h, −80 °C overnight and then transferred into liquid nitrogen (LN₂) containers. The cells stored at 20th and 40th passage were revived after 2 months of storage. Briefly, the frozen cells were thawed quickly at 37 °C and added drop-wise to 15 mL complete medium in a centrifuge tube. The cells were centrifuged at 825 g at 4 °C and the pellet was resuspended in seven mL of complete medium. Cell viability was checked with a hemocytometer following trypan blue staining. The revived cells were seeded into 25 cm² flask and incubated at 28 °C.

Transfection assays

The FtGF cells at the 25th passage (revived from cryopreserved cells) were harvested and grown on coverslip for determining the nature of the cells and to know the transfection efficiency of the cells. The protocols earlier described by Swaminathan et al. (2016b) were followed for immunophenotyping of cells and transfection studies. FtGF cells in 20 fields were counted randomly and the percentage of positive green fluorescent protein (GFP) cells was calculated.

Molecular characterization of the cell line

DNA isolation was carried out by salting out method following Miller, Dykes & Polesky (1988). The DNA was extracted from FtGF cells at 40th passage and muscle tissue of goldfish. PCR was carried out as per Swaminathan et al. (2016b). PCR products of 562 bp and 642 bp were amplified for mitochondrial 16S rRNA and cytochrome c oxidase subunit I (COI) genes using universal primers (F 5′CGC CTG TTT ATC AAA AAC AT 3′ and H 5′CCG GTC TGA ACT CAG ATC ACG T 3′ (Ward et al., 2005) and F 5′TCA ACC AAC CAC AAA GAC ATT GGC AC 3′ and R 5′TAG ACT TCT GGG TGG CCA AAG AAT CA3′ (Palumbi et al., 1991) respectively. The PCR products of both the fragments were sequenced in an ABI 3730 DNA analyzer (Applied Biosystems, Forster City, CA, USA). The sequences of both the mt DNA gene PCR fragments were compared with the published and known sequences in the National Centre for Biotechnology Information (NCBI) database by the basic local alignment search tool (BLAST) (Altschul et al., 1990).

Detection of Mycoplasma contamination in FtGF cell line

Mycoplasma contamination was tested by PCR with 15th and 40th passage FtGF cells grown for 4 days in L-15 medium without antibiotics. Briefly, the harvested cells were centrifuged at 200 g for 10 min and the supernatant was transferred into micro-centrifuge tubes and centrifuged at 250 g to remove debris. The Mycoplasma contamination was checked using EZdetect PCR Kit (HiMedia, Sundara Nagar, Mathikere, Bengaluru) based on amplification of spacer region between 16S rRNA and 23S rRNA. The amplification products were analyzed in 1.5% agarose gel.

CyHV-2 isolation on FtGF cells and viral titer determination

Diseased goldfish obtained from a farm in Kerala, India were confirmed to be infected with CyHV-2 by PCR as per Jeffery et al. (2007). External examination of moribund goldfish revealed clinical signs including simple loss of scales, pale mucous covered gills. Internally, all visceral organs of affected fish were congested and enlarged and white nodules were found on the spleen and the kidney. Gill, kidney and spleen tissues from diseased fish were collected aseptically, and then homogenized in Dulbecco’s Phosphate Buffered Saline (DPBS), freeze-thawed for three cycles and the tissue homogenate was centrifuged at 3,000 g for 30 min at 4 °C. The supernatant was then filtered through a 0.22 µm filter (Millipore) and checked for bacterial contamination before use. Thereafter, 500 µl of the filtered tissue homogenate was inoculated onto 25 cm² flasks after removing the medium and incubated in a shaking incubator (45 rpm) at 28 °C. In control flasks, maintenance medium was used in place of tissue homogenate. After 1 h adsorption, 6.5 mL of the maintenance medium (L-15 medium with 2% FBS) was added to the FtGF flasks, which were incubated at 28 °C. The inoculated flasks were checked daily for cytopathic effects (CPE). The cells from flasks exhibiting 80–90% CPE were harvested along with supernatant. This cell suspension was frozen at −80 °C for further use. Further, to know the temperature range for CyHV-2 replication, FtGF cells were infected with CyHV-2 and incubated at 18, 20 and 25 °C. For titration, FtGF cells were grown in a 96-well plate with a confluence of 70–80%. After removing the medium, filtered tissue homogenate was diluted ten-fold (10⁻¹–10⁻⁹), and 0.1 mL of diluted filtrate was inoculated in triplicate and allowed to adsorb for 1 h. In control wells, DPBS was added in place of filtered tissue homogenate. Thereafter, 0.2 mL of maintenance medium was added to each well and the plate was incubated at 28 °C. The wells were examined daily for the appearance of CPE up to 2 weeks. The virus titer was determined by 50% tissue culture infective dose (TCID₅₀) assay using Reed & Muench (1938) calculations. The viral susceptibility and titration assays of FtGF cells were determined based on three independent experiments.

Experimental challenge studies on goldfish using CyHV-2 propagated in FtGF cells

Clinically healthy goldfish (12–15 cm; 16–23 gm), procured from a local ornamental fish farm, were divided into two groups of 30 fish each and acclimatized in the laboratory aquarium tanks for a week. The tissues from randomly collected goldfish (n = 5) were screened for CyHV-2 using PCR following Jeffery et al. (2007). The fish were anesthetized with MS-222 (Sigma-Aldrich, St. Louis, MO, USA). Fishes in the infected group were challenged with an intraperitoneal (IP) injection of 0.5 mL of 10th passage CyHV-2 FtGF cell culture supernatant, whereas fish in the control group were injected with 0.5 mL maintenance medium. The water temperature was maintained at 28 °C during the experiment and fish were observed daily for clinical signs and mortality. Three goldfish each in the infected and control group were selected randomly after 7 days post-injection (dpi) and screened for CyHV-2 by PCR assay. The experimental challenge study in fish was carried out following ARRIVE guidelines and carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory animals. The experimental challenge trials were evaluated and approved by Institute Animal ethics Committee (IAEC) of ICAR National Bureau of Fish Genetic Resources (NBFGR) vide approval Number G/IAEC/2019/1 dated 04th October 2019.

Confirmation of the CyHV-2 virus

Primary cell culture and maintenance

The primary culture of fin cells from goldfish was achieved using L-15 medium supplemented with 20% FBS at 28 °C. The cells were seen emerging from the sides and edges of caudal fin tissues (Fig. 1A) and they readily attached to the bottom of the flask. A monolayer culture was obtained within 10 days at 28 °C. The monolayer was passaged at 1:2 ratio every 6–7 days. The growth pattern of lag phase (1–3 days), log phase (3–5 days) and stationary phase (5–7 days) was observed. During the initial passages, mixed fibroblast-like and epithelial-like cells were observed and after 10 passages, epithelial cells dominated over fibroblastic cells (Fig. 1B). The FtGF cells have been subcultured for over 55 passages and the cell line has been designated as Fantail Goldfish Fin (FtGF) cell line. The morphology of FtGF cells at 25th passage and at 56th passage are shown in Figs. 1C and 1D.

Characterization of FtGF cell line

The FtGF cells were characterized to determine the ability to grow at different temperatures and different concentrations of FBS, to check the chromosome number of the cells, to authenticate the species origin by DNA barcoding, to examine for the Mycoplasma contamination, to confirm the nature of the cells by immunofluorescence test and to check the transfection efficiency of the cells. The most favorable conditions for culturing of FtGF cells were tested at different incubation temperatures including 20, 24, 28, 35 and 37 °C as well as at different concentrations of FBS viz., 5, 7.5, 10, 15 and 20%. The cells incubated at 20 and 37 °C showed rounding and started detaching on 2nd day. Cells grew in the flask at 24 °C, but the growth rate was slow. Maximum growth of the cells was observed at 28 °C. At 35 °C, FtGF cells proliferated very fast initially, but became puffy and were dying by the 2nd day. Culture of FtGF cells grew best in L-15 with 20% FBS and a slower growth was recorded with decreasing concentration of FBS. The growth kinetics of FtGF cells did not show much difference in 10, 15 and 20% FBS, but cells in 7.5 and 5% FBS had slower growth. The suitable temperature and FBS concentration for the culture of FtGF cells were found to be 28 °C and 10% FBS, respectively (Figs. 2A and 2B). The growth curve of FtGF cells at passage 25 (Fig. 2C) showed as typical “S” shape and the cells were at latent stage before day 1 after seeding and then the cells proliferated rapidly and went into logarithmic stage from day 1 to day 5. The cell number remained stationary between day 5 and day 8, but began to decline after day 9. The FtGF cells grew and proliferated at a steady rate, and their doubling time was calculated to be 33.9 h at passage 25. The FtGF cell growth was arrested in metaphase using Colchicine at final concentration of one µg/mL. Karyotyping results obtained by counting 100 spreads at metaphase showed 82–114 chromosomes, with a distinct peak for the cell line at 104 diploid chromosomes at 30th, 40th and 56th passage. The majority of the cells (60%) had a diploid chromosome number (2n = 104) at all three passage levels. The diploid karyotype of FtGF cells and its frequency distribution at passage 30 are shown (Figs. 3A and 3B) respectively. The FtGF cells at 20th and 40th passage were revived and these exhibited 70–75% viability. The cells were incubated at 28 °C and a monolayer was established within 15 days. Green fluorescent signals were observed in FtGF cells transfected with 2 μg of pAcGFP1-N1 expression vector and strong green fluorescent signals were detected at 52 h post transfection and transfection efficiency was calculated about 28% thus confirming their potential to be used in gene expression studies. (Fig. 3C). The origin of FtGF cell line was confirmed by partial amplification and sequencing of 16S rRNA and COI genes from FtGF cells and goldfish muscle and comparison with available COI and 16S rRNA sequences in GenBank. Nucleotide sequence analysis revealed 100% similarity with sequences from goldfish muscle and maximum similarity (99%) with COI (Accession number KX145542 and KX145499) and 16S rRNA sequences (Accession number AJ247070, KY231826) of goldfish in GenBank. These data confirm that the origin of the developed FtGF cell line is from goldfish. No target band of spacer region between 16S and 23S rRNA of Mycoplasma was observed indicating that the FtGF cells were free of Mycoplasma contamination.

CyHV-2 isolation on FtGF cells and virus titration assays

FtGF cells were infected with CyHV-2 and evaluated by significant CPEs observed. In this study, CyHV-2 was propagated with high viral titer in the FtGF cell line. The FtGF cell line (Fig. 4A) infected with CyHV-2 began to show morphological changes from the 2nd dpi (Fig. 4B) and the CPE such as cell elongation, rounding and cell fusion with cytoplasmic vacuolation were observed from 4th dpi (Fig. 4C) at 28 °C. Cell death started from the 6th dpi (Fig. 4D) and complete detachment was observed by 10 days. The appearance of typical CPE in FtGF cells infected with Indian CyHV-2 was observed at 8 dpi, 6 dpi and 5 dpi when the cells were incubated at 18, 20 and 25 °C respectively after inoculation of the virus, and it was observed that the efficient propagation of Indian CyHV-2 was most rapid (2 dpi) at 28 °C. The infected FtGF cells and the supernatant were confirmed for the presence of CyHV-2 by PCR and sequencing. No CPE was observed in the control cells inoculated with maintenance medium. The TCID₅₀ of the culture suspension harvested from infected FtGF was found to be 10^{7.8 ± 0.26} TCID₅₀/mL (Fig. 5A). The CyHV-2 could be propagated in FtGF flasks for 20 passages.

Virulence of CyHV-2 propagated in FtGF cells to goldfish

To confirm the infectivity of CyHV-2 propagated on FtGF cell line, an experimental challenge study was carried out in healthy goldfish. The goldfish challenged with 1.4 × 10⁷ TCID₅₀/mL CyHV-2 (passage 10) produced in the FtGF cell line began to die at 5 days post inoculation (dpi) and the mortality reached 100% at 12 dpi (Fig. 5B). The dead fish from infected groups exhibited similar clinical signs seen in naturally infected fish. The clinical signs of challenged goldfish were hemorrhages on the body surface, exophthalmia, pale gills and a swollen abdomen (Figs. 5C and 5D). There was no mortality in the mock-infected group. The tissues from randomly selected fish from the infected groups tested positive for CyHV-2 by PCR and produced CPE in FtGF, while fish from control group were negative. The CyHV-2 recovered from experimentally infected fish was also found to be virulent to goldfish and caused similar symptoms (T. Swaminathan, 2020, unpublished data).

Confirmation of the CyHV-2 virus

The confirmation of the CyHV-2 infection in infected tissues, FtGF cell culture suspension, and experimentally challenged fish was carried out by amplification of fragment of DNA polymerase gene using PCR, histopathological investigation and transmission electron microscopy.

Polymerase chain reaction

Tissue samples (gills, spleen and kidney) collected from naturally infected tissues, FtGF cell culture suspension, and experimentally challenged fish were found to be positive for CyHV-2 in PCR assay. The expected PCR product of 362 bp was obtained in all CyHV-2 positive samples. A GenBank BLAST search on the sequence revealed a high identity to CyHV-2 isolate SYC1 strain (KM200722, 99.9%) and CyHV-2 isolate STJ1 strain (JQ815364, 99.9%). The sequence of the DNA polymerase gene of CyHV-2 isolated from goldfish in this study was deposited in GenBank (GenBank accession nos. KU527548 and KU527549).

Histopathology

Hypertrophied nuclei with marinated chromatin material in sections of gills, spleen and kidney from goldfish experimentally infected with CyHV-2 (Figs. 6A–6C) and inflammatory exudate between the secondary lamellae were observed in sections of gills. The lesions of the experimentally challenged fish were compared with the sections of gills, spleen and kidney in naturally CyHV-2 infected goldfish.

Transmission electron microscopy

The mature virus particles were observed ultra structurally in cytoplasm of cells in gills as well as spleen tissue of experimentally challenged fish (Figs. 7A and 7B) and CyHV-2 infected FtGF cells that were about 170–180 nm in diameter (Fig. 7C). Supernatant of FtGF cells with CPE were examined by TEM, and viral particles morphologically similar to a herpesvirus were observed (Fig. 7D).

Ethical statement

All the experimental challenge procedures in this study (Proposal number: NBFGR/IAEC/2019/0014) were evaluated and approved by Institute Animal ethics Committee (IAEC) of ICAR National Bureau of Fish Genetic Resources (NBFGR) (CPCESA Registration No: 909/GO/Re/S/05/CPCSEA dated 09.09.2005 and CPCSEA Ref file No. 25/111/2014-CPCESA dated 05th December 2018) vide approval Number G/IAEC/2019/1 dated 04th October 2019.