Methods of producing viruses

Abstract

Methods of producing, propagating and multiplying viruses, methods of identifying the presence of a virus in a sample, specifically methods using Japanese eel cell cultures. Also provided are an isolated Japanese eel cell culture, optionally infected with a virus, vaccines comprising viruses grown according to the methods of the invention, and a kit for propagating viruses comprising the Japanese eel cell culture. The virus may be a fish virus such as nodavirus, Infectious Pancreatic Necrosis Virus (IPNV) and Salmon Pancreas Disease Virus (SPDV).

Claims

1. A method of propagating a virus comprising the steps: infecting a cell culture with the virus; culturing the infected cell culture; harvesting the virus; and optionally purifying the virus, wherein said cell culture is the cell culture Designated Anguilla japonica-K deposited with accession number 16062701.

2. The method according to claim 1, wherein the virus is a fish virus.

3. The method according to claim 1, wherein the virus is one selected from the group consisting of: nodavirus; Infectious Pancreatic Necrosis Virus (IPNV); and Salmon Pancreas Disease Virus (SPDV).

4. The method according to claim 1, wherein the cell culture is infected with viral stock at a Multiplicity of Infection (MOI) of between 0.1 and 10.

5. The method according to claim 1, wherein the virus is harvested at a final titre of the between about 210.sup.6 TCID.sub.50/ml and about 210.sup.11 TCID.sub.50/ml.

6. The method according to claim 1, wherein the virus is harvested when a cytopathic effect (CPE) is observed in the cells.

7. The method according to claim 1, wherein the virus is harvested from a supernatant and/or lysed cells.

8. An isolated Japanese eel cell from the cell culture designated Anguilla japonica-K deposited with accession number 16062701 infected with a virus.

9. The isolated Japanese eel cell according to claim 8, wherein the Japanese eel cell is from the cell culture designated Anguilla japonica-K deposited with accession number 16062701.

10. An isolated Japanese eel cell according to claim 8, wherein the virus is one selected from the group consisting of: nodavirus; Infectious Pancreatic Necrosis Virus (IPNV); and Salmon Pancreas Disease Virus (SPDV).

11. An isolated cell culture designated Anguilla japonica-K deposited with accession number ECACC 16062701.

Description

(1) The present invention is described with reference to the accompanying drawings in which:

(2) FIG. 1 shows the results of size separation by agarose gel electrophoresis of the products of PCR analysis of the Anguilla japonica-K cell culture deposited under ECACC accession number 16062701;

(3) FIG. 2 is a graph showing the growth curve for Infectious Pancreatic Necrosis Virus isolate IPN NVI 015 (GenBank accession numbers AY379740 and AY379741) in Anguilla japonica-K cell culture (ECACC accession number 16062701) in terms of the virus titre at 5, 6, 7 and 8 days post-harvest, infected at an MOI of 1;

(4) FIG. 3 is a graph showing the growth curve for Salmon Pancreas Disease Virus (SPDV) in Anguilla japonica-K cell culture (ECACC accession number 16062701) in terms of the virus titre at 5, 6, 7, 12 and 14 days post-harvest, infected at an MOI of 0.5; and

(5) FIG. 4 is a graph showing the growth curve for Nodavirus in Anguilla japonica-K cell culture (ECACC accession number 16062701) in terms of the virus titre at 4, 5, 6, 7, 9 and 11 days post-harvest, infected at an MOI of 0.1, 0.5 and 1.

EXAMPLES

Example 1

Species Identity of the Anguilla japonica-K Cell CultureMolecular Testing

(6) Aliquots of Anguilla japonica-K pre-master cell seed at passage (p) 118 and Anguilla japonica-K master cell seed at p 121 were tested for species identity by polymerase chain reaction (PCR) and sequencing.

(7) Materials and Methods

(8) Genomic DNA was extracted from aliquots of the master cell seed p121 and pre-master cell seed p118 in duplicate using an EZ1 DNA tissue kit and EZ1 Biorobot (Qiagen) and were subjected to PCR amplification with primers for the mitochondrial cytochrome oxidase 1 (CO1) and 16S genes (Table 1 below). Products were visualised after size separation by agarose gel electrophoresis (FIG. 1). All products were excised, purified and sequenced directly by cycle sequencing (BigDye Terminator v3.1 Cycle sequencing kit and ABI 3130xl Avant genetic analyser (Applied Biosystems)). The consensus sequences from the duplicate analyses were compared to published sequences available in GenBank and EMBL by BlastN analysis.

(9) TABLE-US-00001 TABLE1 Fragment Gene size Name Primersequences CO1 <650bp FishF1 5-TCAACCAACCACAAAGACATTGGCAC-3 FishR1 5-TAGACTTCTGGGTGGCCAAAGAATCA-3 16S <410bp 16SarL 5-CGCCTGTTTATCAAAAACAT-3 16SbrH 5-CCGGTCTGAACTCAGATCACGT-3
Results

(10) FIG. 1 shows the Amplification products for CO1 gene (left hand set, lanes 1-4) and 16S gene (right hand set, lanes 7-10) of pre master Anguilla japonica-K cell seed p118 (1&2 and 7&8 and master cell p121 (3&4 and 9&10). Lanes 5, 6, 11 and 12 are negative controls.

(11) PCR and sequencing of the CO1 and 16S genes of aliquots of pre-master cell seed at p118 and master cell seed at p121 all show 100% identity to the respective genes of Japanese eel (Anguilla japonica). There was no evidence of mixed sequence.

(12) The Anguilla japonica-K cell culture is of A. japonica origin.

Example 2

Propagation of Anguilla japonica-K Cells

(13) Methods and experimental protocols for growing cells and cell cultures would be known to persons skilled in the art. Some examples of methods according to the present invention are described below.

(14) Cell Storage Conditions

(15) The cell culture was stored in 50/50 by volume of 10% DMSO in Minimal Essential Medium (MEM) and Foetal Bovine Serum (FBS) undiluted, at a density of 210.sup.6 cells/ml.

(16) Cell Culture Conditions

(17) Anguilla japonica-K cells were generally maintained at 28 C. with L-15 medium (Invitrogen) supplemented with 10% L-glutamine and 1 L/mL of gentamicin, and 7.5% Foetal Bovine Serum (FBS).

(18) Alternatively, the Anguilla japonica-K cells were maintained at between 15 and 28 C. in Minimal Essential Medium (MEM), 7.5% Foetal Bovine Serum (FBS), 0.5% sodium bicarbonate, and 1% L-alanyl-L-Glutamine dipeptide (Glutamax).

(19) Anguilla japonica-K cells infected with IPN were maintained at 15 C. Anguilla japonica-K cells infected with nodavirus were maintained at 28 C.

(20) Passage

(21) The cells were passaged at a seeding ratio of 1:6, or cell seeding rate of 1.610.sup.4 cell/cm.sup.2.

Example 3

Anguilla japonica-K Cell Culture, ECACC Accession Number 16062701

(22) The Anguilla japonica-K cell culture deposited with the ECACC relates to a sample of the pre-master cell seed at passage (p) 124. Analysis showed the cell culture to be mainly epithelial, with fibroblasts present.

Example 4

Assessment of Viral Yields

(23) Approximately 80% confluent Anguilla Japonica-K cells maintained in L15 media with 2% FBS and at a density of 610.sup.4 cells/cm.sup.2 were inoculated with a virus (Infectious Pancreatic Necrosis (IPN), Salmon Pancreas Disease Virus (SPDV) or Nodavirus), followed by incubation at 15 C.

(24) The cells were inoculated at a MOI of 1.0 for IPN and SPDV, and MOI's of 0.1, 0.5 and 1.0 for Nodavirus.

(25) The growth curves for the viruses are shown in FIG. 2 (Infectious Pancreatic Necrosis Virus), FIG. 3 (Salmon Pancreatic Disease Virus) and FIG. 4 (Nodavirus).

(26) Larger scale productions of the three viruses (IPN, SPDV and Nodavirus) in cell factories were also assessed and the virus titres at optimal harvest times were recorded. Cells were infection at an MOI of 1 for IPN, an MOI of 0.5 for SPDV and an MOI of 0.1 for Nodavirus.

(27) The virus was harvested following full CPE by centrifugation of the suspension at 2500g followed by recovery of the supernatant. The average titres of the viruses are shown in Table 2 below.

(28) TABLE-US-00002 TABLE 2 Average titre Optimal (TCID.sub.50/ml) at harvest optimal harvest time time in Virus grown in over several research Number of days post- Anguilla japonica-K scale batches (cell batches (cell infection cells factories) factories) (days) SPDV .sup.6.6 10.sup.9 5 ~15 Nodavirus 4.65 10.sup.10 2 7-11 IPN 1.58 10.sup.10 1 8-12