Anti-Birnavirus Activity of Methisoprinol – i n vitro Study with Infectious Pancreatic Necrosis Virus ( IPNV )

Siwicki A. K. , M. Morand, F. Pozet , B. Kazun: Anti-Birnavirus Activity of Methisoprinol – in vitro Study with Infectious Pancreatic Necrosis Virus (IPNV). Acta Vet. Brno 2002, 71: 543-547. This study was conducted to evaluate the influence of methisoprinol, synthetic anti-viral product, on the IPNV replication in vitro by measuring viral RNA synthesis. The monolayers of RTG-2 cells in tissue culture plates (Multiwell, 24 wells, Becton Dickinson, USA) was cultivated with different concentrations of methisoprinol (Polfa, Poland) 0, 100, 200, 400, 500 and 1 000 μg/ml of medium and were followed by infection with 100 μl of IPN virus suspension containing 107 TCID50/ml, in triplicate. At 24, 48 and 72 h after infection, the IPN virusinfected and methisoprinol-applicated RTG-2 cell cultures were submitted to one hour starvation and after two hours incubation with 10 μCi/ml of [3H]-uridine. Culture homogenates of each isolate were incubated with phenol/chloroform to extract RNA and followed by slab polyacrylamide gel electrophoresis for 2 h. The gel strips were dissolved and the counts per minute (cpm) evaluated in a scintillation counter. The replicative cycle of IPN virus in RTG-2 cell culture was rapid. In control group (only infected by IPNV), the incorporation of [3H]uridine was 45 000 ± 1 500 cpm at 24 h, 186 000 ± 2 450 cpm at 48 h and 554 500 ± 4 550 cpm at 72 h. The percent of inhibition of IPN viral RNA labelling under methisoprinol application ranged from 5% at 24 h to 85% at 72 h depending on concentration of tested product and time when cultures were harvested. The highest percent of inhibition at 72 h after infection was observed at the dose 1000 μg/ml. The results of these experimental studies show the inhibition of incorporation (cpm) of [3H]-uridine into IPN viral RNA in cell cultures exposed with methisoprinol at various concentrations. Fish, IPNV, methisoprinol, replication The family Birnaviridae presents the smallest RNA viruses and consists of three genera: Aquabirnavirus, Avibirnavirus and Entomobirnavirus. The aquatic birnaviruses are the largest group of viruses including several strains from different fish species and invertebrates. The Birnaviridae are the most extensively studied fish viruses, because they are ubiquitous in aquatic organisms, since they have been isolated all over the world from both freshwater and marine fish of different species, and are responsible for severe losses in aquaculture. Infectious pancreatic necrosis virus (IPNV), the genus Aquabirnavirus, is the prototype of the birnavirus infecting fish, molluscs and crustaceans. The singleshelled, naked, icosahedral viral particles of birnaviruses containing 2 segments (A and B) have double-stranded RNA. Their dsRNA genome is easily purified and is resistant to common RNase. Very little is known about the replication strategy of birnaviruses. A viral protein 1 (VP1) is found in the viral particle both as a free form and bound to the RNA genome-linked protein (VPg) and is thought to be the viral ACTA VET. BRNO 2002, 71: 543–547 Address for correspondence: Prof. A.K. Siwicki, DVM, PhD Dept. of Microbiology and Clinical Immunology Faculty of Veterinary Medicine University in Olsztyn, 10-917 Olsztyn-Kortowo, Poland Phone: + 48 22 756 24 90 Fax: + 48 22 756 24 90 e-mail: aksiw@infish.com.pl http://www.vfu.cz/acta-vet/actavet.htm

This study was conducted to evaluate the influence of methisoprinol, synthetic anti-viral product, on the IPNV replication in vitro by measuring viral RNA synthesis.The monolayers of RTG-2 cells in tissue culture plates (Multiwell, 24 wells, Becton Dickinson, USA) was cultivated with different concentrations of methisoprinol (Polfa, Poland) 0, 100, 200, 400, 500 and 1 000 µg/ml of medium and were followed by infection with 100 µl of IPN virus suspension containing 10 7 TCID 50 /ml, in triplicate.At 24, 48 and 72 h after infection, the IPN virusinfected and methisoprinol-applicated RTG-2 cell cultures were submitted to one hour starvation and after two hours incubation with 10 µCi/ml of [ 3 H]-uridine.Culture homogenates of each isolate were incubated with phenol/chloroform to extract RNA and followed by slab polyacrylamide gel electrophoresis for 2 h.The gel strips were dissolved and the counts per minute (cpm) evaluated in a scintillation counter.The replicative cycle of IPN virus in RTG-2 cell culture was rapid.In control group (only infected by IPNV), the incorporation of [ 3 H]uridine was 45 000 ± 1 500 cpm at 24 h, 186 000 ± 2 450 cpm at 48 h and 554 500 ± 4 550 cpm at 72 h.The percent of inhibition of IPN viral RNA labelling under methisoprinol application ranged from 5% at 24 h to 85% at 72 h depending on concentration of tested product and time when cultures were harvested.The highest percent of inhibition at 72 h after infection was observed at the dose 1000 µg/ml.The results of these experimental studies show the inhibition of incorporation (cpm) of [ 3 H]-uridine into IPN viral RNA in cell cultures exposed with methisoprinol at various concentrations.

Fish, IPNV, methisoprinol, replication
The family Birnaviridae presents the smallest RNA viruses and consists of three genera: Aquabirnavirus, Avibirnavirus and Entomobirnavirus.The aquatic birnaviruses are the largest group of viruses including several strains from different fish species and invertebrates.The Birnaviridae are the most extensively studied fish viruses, because they are ubiquitous in aquatic organisms, since they have been isolated all over the world from both freshwater and marine fish of different species, and are responsible for severe losses in aquaculture.
Infectious pancreatic necrosis virus (IPNV), the genus Aquabirnavirus, is the prototype of the birnavirus infecting fish, molluscs and crustaceans.The singleshelled, naked, icosahedral viral particles of birnaviruses containing 2 segments (A and B) have double-stranded RNA.Their dsRNA genome is easily purified and is resistant to common RNase.Very little is known about the replication strategy of birnaviruses.A viral protein 1 (VP1) is found in the viral particle both as a free form and bound to the RNA genome-linked protein (VPg) and is thought to be the viral RNA dependent RNA polymerase (Duncan and Dobos 1986;Duncan et al. 1987;Duncan et al. 1991).
IPNV has been detected for years in several places of the world with high mortality rates of rainbow trout fry at the temperatures of 8-12 o C.This virus is spread by shipment of contaminated fish eggs and it is probably present in all trout-farming countries.Actually, one of the very important questions is to develop some prevention methods against IPNV infection (Okam oto et al. 1993).Protection of fish against viral diseases by vaccine has been developed without relevant success.Therefore, research is concentrated on how to improve the anti-viral protection and develop some anti-viral products to selectively inhibit virus replication (Bradshaw and Sumner 1977;Ginsberg and Glasky 1977;Berkman et al. 1979;Ballet et al. 1982;Zagni and Cannarozzo 1982;Linhares et al. 1996).Methisoprinol is a synthetic compound formed from the p-acetamidobenzoate salt of N-N dimethylamino-2-propanol and inosine in a 3:1 molar ratio.The in vitro and in vivo studies show antiviral and antitumour activities of methisoprinol secondary to an immunomodulating influence on cell-mediated immunity and anti-viral immune responses (Delogu et al. 1982;Fudenberg and Whitten 1984).Methisoprinol has been shown to act in vitro and in vivo by inhibiting the replication of various single-stranded and doublestranded RNA-containing viruses (Chang and Weinstein 1973;Linhares et al. 1989;Linhares et al. 1996).In our preliminary in vitro study, we demonstrated that methisoprinol at different concentrations inhibited the replication of the two salmonid rhabdoviruses VHSV and IHNV (Siwicki et al. 2002).
In this study, we continue to examine the in vitro influence of methisoprinol on the infectious pancreatic necrosis virus (IPNV) replication by measuring viral RNA synthesis.

Materials and Methods
The cell line, rainbow trout gonad (RTG-2) was used for the isolation, propagation and identification of IPN virus.RTG-2 cell line was propagated in Glasgow medium (BHK21 Medium, Life Technologies), buffered to pH 7.45 with 0.16 M Tris-HCL (Sigma) and supplemented with 10% foetal calf serum (FCS, Life Technologie) and antibiotics (penicillin 100 IU/ml, streptomycin 0.1 mg/ml and kanamycin 0.1 mg/ml).IPN virus isolated from diseased fish from a French trout farm in Laboratoire Veterinaire Departemental (LDA39) Lons le Saunier (Siwicki et al. 1998) were propagated in RTG-2 cell line.After infection, the RTG-2 cells were incubated at 15 o C in medium and supplemented with 2% FCS and antibiotics (penicillin 100 IU/ml, streptomycin 0.1 mg/ml and kanamycin 0.1 mg/ml).The virus was harvested when the cytopathic effect was complete and quantified by titration against RTG-2 cells in 96-well microtitre plates in tissue culture infective doses of 50%/ml (TCID 50 ).
Methisoprinol (series 33-03-00; Polfa Grodzisk, Poland) was used in this study.The stock solution was prepared in medium (Glasgow MEM) at the concentration 2 000 µg/ml and stored at 4 0 C for not more than 3 days.
The monolayers of RTG-2 cells in tissue culture plates (Multiwell, 24 wells, Becton Dickinson, USA) were cultivated with 0.75 ml of maintenance medium, free of FCS, containing 0, 100, 200, 400, 500 and 1 000 µg/ml methisoprinol.The cell cultures containing different concentration of methisoprinol were followed by infection with 100 µl of IPN virus suspension containing 10 7 TCID 50 /ml, in triplicate.The negative control (without infection by IPNV) was used in each concentration of methisoprinol.At 24, 48 and 72 h after infection, the IPN virus-infected and methisoprinol-applicated RTG-2 cell cultures were submitted to one hour starvation period by replacing medium with pre-heated PBS, at pH 7.4, followed by a two-hour incubation with 10 µCi/ml of [ 3 H]-uridine (27 Ci/mmol; Amersham Int., UK) in maintenance medium, according to the method presented by Linhares et al. (1996).Cultures were submitted to three freezing and thawing cycles.Culture homogenate of each isolate were incubated with phenol/chloroform to extract RNA, followed by slab polyacrylamide gel electrophoresis for 2 h at 25 mA, using a 7% separation polyacrylamide gel.The silver nitrate staining was used for the detection of RNA bands.The gel strips were dissolved in a solution containing 30 % hydrogen peroxide and 0.9 N ammonium hydroxide, according to the method presented by Bonner and Laskey (1974).After, the scintillation fluid for aqueous samples (Sigma Chemicals, USA) was added and the counts per minute (cpm) evaluated in a scintillation counter (Beckman LS).
Statistical analyses were performed using the Student's t-test.Data are reported as means ± SD for 4 experiments in triplicate as percent inhibition of virus RNA synthesis by methisoprinol compared to infected RTG-2 cells not exposed with methisoprinol.

Results and Discussion
In the present study, we continued the in vitro experiments to examine the influence of different concentrations of methisoprinol on the replication of infectious pancreatic necrosis virus, aquatic birnavirus, responsible for high mortality rates of trout fry and fingerlings in all major trout-farming countries.We examined the anti-viral influence of methisoprinol by determination of the inhibition the viral RNA synthesis at the 24, 48 and 72 h after infection.Monitoring the influence of different concentrations of methisoprinol on the synthesis of IPN virus RNA in RTG-2 cells culture by radioisotope labelling permitted the quantitative determination of the inhibition and provided information about the early stage of infection since the method permitted the detection of minute quantities of viral RNA.The replicative cycle of IPN virus in RTG-2 cell culture was rapid.In control group, only infected by IPN virus, the counted level after incorporation of [ 3 H]-uridine was 45 000 ± 1500 cpm at 24 h, 186 000 ± 2450 cpm at 48 h and 554 500 ± 4550 cpm at 72 h.However, a cytopathic effect was observed in virus-infected cultures without methisoprinol (control) at 24 h after infection.Cell cultures exposed only to the methisoprinol did not show any morphological change to the end of experimental time.IPNV-infected culture exposed with methisoprinol at the concentration of 100 and 200 µg/ml showed a small (maximum 25% at dose 100 µg/ml and about 10% at dose 200 µg/ml) cytopathic effect and no cytopathic effect was observed in cultures treated with 400, 500 and 1 000 µg/ml of the methisoprinol.Fig. 1 presents the in vitro influence of different concentrations of methisoprinol on the percent of inhibition of IPNV RNA synthesis compared to infected cells not exposed on methisoprinol at 24, 48 and 72 h after infection of RTG-2 cells line.The percent of inhibition of IPN viral RNA labelling under methisoprinol application ranged from 5% at 24 h to 85% at 72 h depending on concentration of tested product and time when cultures were harvested.The highest percent of inhibition at 72 h after infection was observed at the dose of 1000 µg/ml.
The results of this experimental study shows the inhibition of incorporation (cpm) of [ 3 H]uridine into IPN viral RNA in cell cultures exposed with methisoprinol at various concentrations.A low level of incorporation occurred at 24 h after infection, with a non-significant rate of viral replication in methisoprinol-exposed cultures, which was comparable to that of unexposed cultures.The percent of inhibition of viral RNA at 24 h after infection was low, probably because of the low incorporation of [ 3 H]-uridine as a result of the non-significant quantity of viral RNA synthesized.These results are in agreement with the study with other fish viruses.The results of experimental study show the inhibition of incorporation of [ 3 H]-uridine into VHS and IHN viral RNA in cell cultures exposed to methisoprinol at various concentrations (Siw icki et al. 2002).The highest percentage of inhibition of viral RNA was observed 72 h after exposition to the dose of 500 g/ml of medium.In tissue cultures methisoprinol has been reported to inhibit the replication of several RNA and DNA viruses in animals (Chang and Weinstein 1973;Ginsberg and Glasky 1977;Hernandez-Jauregui et al. 1980;Pompidou et al. 1985;Tsang and Fudenberg 1982;Zagni and Cannarozzo 1982;Linhares et al. 1996).Linhares et al. (1996) observed also a very low percentage of inhibition of rotavirus 24 h post infection and suggested that this observation is a result of the non-significant quantity of viral RNA synthesis.
In our in vitro study we observed the anti-viral activity of methisoprinol, which at different concentrations, inhibits the IPN virus replication.The results suggested that additional in vivo study must be carried out for the possibility of application of methisoprinol in prevention or therapy of IPNV infection in intensive fish culture.

Fig. 1 .
Fig.1.The in vitro effect of different concentrations of methisoprinol on the percent of inhibition of IPNV RNA synthesis compared to infected cells not exposed to methisoprinol at 24 h, 48 h and 72 h after infection of RTG-2 cells (mean SD, n=12)