In vitro antiviral activity and kinetics of the inhibitory effect of some compounds against Feline calicivirus strain F 9 – a surrogate model of human noroviruses

© 2018 Yulian Dimitrov Tumbarski. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercialShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/). *Corresponding Author Yulian Dimitrov Tumbarski, Department of Microbiology, University of Food Technologies, 26, Maritsa Blvd., 4002, Plovdiv, Bulgaria. E-mail: tumbarski @ abv.bg In vitro antiviral activity and kinetics of the inhibitory effect of some compounds against Feline calicivirus strain F9 – a surrogate model of human noroviruses

Genera Norovirus and Sapovirus include human enteric viruses.Members of these genera are responsible for sporadic cases and epidemic outbreaks of acute gastroenteritis in humans.
Noroviruses are recognized as one of the leading etiological agents of acute viral gastroenteritis among the people of all ages in the industrialized countries.In recent two decades noroviruses are estimated to be responsible for over 90% of all outbreaks of acute viral gastroenteritis in Europe and the U.S.A., and for this reason they remain a serious problem of public health (Lopman et al., 2003;Zheng et al., 2010;Hall et al., 2012;Tumbarski et al., 2013;Vega et al., 2014).
Animal caliciviruses are known as causative agents of respiratory illness, conjunctivitis, stomatitis and hemorrhagic disease in animals (Bhella et al., 2008).Genus Vesivirus includes the Feline calicivirus (FCV) which causes acute upper respiratory disease, stomatitis, diarrhea and limping disease in cats (Radford et al., 2007); Vesicular exanthema of swine virus (VESV) associated with the vesicular exanthema in swine and San-Miguel sea lion virus (SMSV) which causes abortions and vesicular lesions in sea lions (Green et al., 2000).Genus Lagovirus includes the Rabbit hemorrhagic disease virus (RHDV) which causes the rabbit hemorrhagic disease and European brown hare syndrome virus (EBHSV) -the European brown hare syndrome in rabbits (McIntosh et al., 2007).Genus Nebovirus contains a single species -the bovine enteric calicivirus associated with enteric diseases in calves (Newbury-1 virus) (Di Martino et al., 2011).A second nebo-like virus (Kirklareli virus) which may represent a second species within this genus has been described in cattle in Turkey (Alkan et al., 2015).
Caliciviruses are small, nonenveloped, positive-sense RNA containing viruses.The viral genome is approximately 7.5 kb in length, encapsidated by an icosahedral capsid, which is 35 to 40 nm in diameter and consisting of 90 VP1 dimers.The genome contains up to four open reading frames (ORFs), the first of which (ORF1) encodes a polyprotein that is post-translationally cleaved to produce several nonstructural proteins critical to virus replication.ORF2 and ORF3 encode the major capsid protein VP1 and a putative minor structural protein VP2, respectively.ORF4 is still with unknown function (Bhella et al., 2008).In cell cultures, FCV strains grow fast and cause rapid cytopathic effects morphologically expressed in rounding and detachment of infected cells from the vessel surface followed by cell death (Ossiboff et al., 2007).
At present, there are not enough data published in the scientific literature for the specific treatment and prevention of the infections caused by caliciviruses, respectively noroviruses.Also little is known about the different antiviral compounds and their potential application as effective means for anticalicivirus chemotherapy.The inability of noroviruses to grow in cell cultures has greatly hampered the development of reliable methods for their detection, viability testing and finding of effective antiviral compounds.Therefore, knowledge of efficient inactivation by different methods or specific antivirals is limited and based on studies with cultivatable surrogate models of human noroviruses such as Feline calicivirus (FCV) (Bidawid et al., 2003;Duizer et al., 2004).
The aim of current study is to evaluate in vitro cytotoxicity, antiviral activity and kinetics of the effect of some highly efficient virus replication inhibitors with a different mode of action against the Feline calicivirus strain F9 -a surrogate model of human noroviruses.

Virus
Feline calicivirus (FCV) strain F9 (kindly supplied by Prof. S. Sattar, Faculty of Medicine, the University of Ottawa, Canada) was used.The molecular mass and the structural formula of the compounds are presented in Table 1.

Compounds
All compounds with exception of ribavirin were preliminary dissolved in dimethyl sulfoxide -DMSO (Fluka AG, Switzerland) and then in maintenance DMEM to the required molar concentrations.

Neutral red uptake assay
Neutral red uptake assay to determine the cytotoxicity and antiviral effect of all compounds was used.Confluent monolayers of CRFK cells in 96-well microplates (Cellstar, Greiner bio-one, Germany) after aspiration of the culture medium were infected with 100 µL per well of three viral inoculation doses -10, 100 and 1000 CCID 50 (excluding the blanks and cell control wells).After 90 min of adsorption at 37°C and 5% CO 2 , the excessive virus was discarded and 100 µL per well of serial dilutions of the tested compounds in DMEM were added.After 3 days of incubation at 37°C and 5% CO 2 , the medium was removed and the cells were washed with 150 µL per well of prewarmed phosphate-buffered saline (PBS), which was aspirated and replaced with 100 µL per well of 0.4% neutral red (NR) (Fluka AG), preliminarily dissolved in DMEM.Cells were incubated for additional 3 hours at 37°C + 5% CO 2 .Then NR was removed, monolayers were washed with PBS and 150 µL per well of NR-desorb solution (acetic acid, ethanol and dd H 2 O -1:50:49) was placed.Plates were shaken for 10 min until NR has been extracted and formed a homogeneous solution.Measurement of the optical density (OD) was determined at λ = 540 nm in ELISA-reader 530 (Organon Teknika, Germany) according to the standard protocol.

One-step virus growth cycle experimental design
One-step virus growth cycle experimental design (timing-of-addition study) was carried out to determine the kinetics of the inhibitory activity of compounds against FCV-F9.Monolayers of CRFK cells were cultured for 24 hours in plastic tubes (Cellstar, Greiner bio-one, Germany).After removing the culture medium, viral inoculum (multiplicity of infection = 10) was placed for adsorption (90 min at 37°C and 5% CO 2 ).The excessive virus was discarded and the cells were washed with Hank's solution.Then 900 µL per tube of maintenance DMEM were placed.The compound was added to DMEM (excluding the virus control tubes) in its maximal effective concentration at 0 th , 1 st , 2 nd , 3 rd , 4 th and 5 th hour after the virus adsorption.The samples were frozen subsequently at 2 nd , 3 rd , 4 th , 6 th and 8 th hour for later titration.The samples were tested in monolayers of CRFK cells for presence/absence of cytopathic effect (CPE) and infectious virus titer was determined by Reed and Muench classical method (1938).

Cytotoxicity assay
Cytotoxicity tests of all compounds were done in the same plates simultaneously with antiviral tests.After formation of the cell monolayer, the growth medium was discarded and 100 µL of serial dilutions of the tested compounds in maintenance DMEM were added.During the incubation for 3 days, monitoring for microscopic cytotoxic effects at 24 th , 48 th and 72 nd hour was carried out.The results were read at the 72 nd hour by the neutral red uptake assay described above.

RESULTS AND DISCUSSION
The 50% cytotoxic concentrations (CC 50 ) of tested antiviral compounds were determined for the CRFK cell line, which was used for propagation of FCV-F9 in all experiments.
As seen from the results presented in Table 2, the compounds ribavirin, HBB, and PTU-23 were relatively non-toxic for the CRFK cell line.In contrast, oxoglaucine was non-toxic only in the lower molar concentration and seemed to have a relatively low value of CC 50 .
The tested antiviral compounds oxoglaucine, ribavirin, HBB, and PTU-23 showed inhibitory activity against FCV-F9 replication in varying degree.The inhibitory effect of compounds depended on their concentration in the culture medium as well as on the dosage of viral inoculum (Table 2).
The antiviral compounds demonstrated the inhibitory effect on FCV-F9 replication in low virus inoculation doses -100 CCID 50 and 10 ССID 50 .At virus inoculation dose 100 ССID 50 , only one compound -HBB did not show anticalicivirus activity.
At virus inoculation dose 10 ССID 50 all tested compounds showed activity against FCV-F9.These data are comparable to our previously published results from the primary screening obtained by the cytopathic effect (CPE) inhibition method (Tumbarski and Galabov, 2008).

Antiviral activity and kinetics of the inhibitory effect of oxoglaucine on the replication of FCV-F9
The recently explored and described aporphinoid alkaloid oxoglaucine isolated from the aerial parts of the plant Glaucum flavum Cranz, showed the highest anticaliciviral activity and selectivity ratio (CC 50 /IC 50 ) -39.85 (Table 2).Some authors reported remarkable inhibitory activity of oxoglaucine also against the replication of human rhinovirus 14 (HRV-14) and a large spectrum of enteroviruses (Georgieva and Galabov, 2008;Nikolaeva-Glomb et al., 2008).
Tablе 3: Antiviral effect of oxoglaucine on the replication of FCV-F9 in onestep virus growth cycle.In one-step virus growth cycle experiments, some characteristics of FCV replication cycle were taken into accountrapid growth, a lag phase of about 3.5 h, followed by exponential growth of about 3-4.5 h and maximal production of virus (peak yield) at 8-12 h post infection (p.i.) (Ossiboff et al., 2007).
Oxoglaucine revealed maximal inhibitory effect when added to the maintenance medium during the first four hours p.i. which coincided with the lag phase of FCV-F9 replication.This led to a significant reduction of the infectious virus titer with 2.6 log CCID 50 /mL at the 4 th h of virus replication compared to the virus control levels.Adding of oxoglaucine during the exponential phase of virus cycle did not affect the virus replication and it reached the peak titre at 8 th h p.i. (Table 3).

Antiviral activity and kinetics of the inhibitory effect of ribavirin on the replication of FCV-F9
According to the antiviral activity and selectivity ratio, the broad-spectrum antiviral agent ribavirin occupied an intermediate position with selectivity ratio of 27.71 vs. virus inoculation dose 10 CCID 50 (Table 2).The antiviral compound ribavirin possesses a polycomponent mechanism of action.Its influence is based predominantly on various effects of the host cells by reduction of GTP pool; inhibition of 5'-cap formation on mRNAs, etc. (Galabov and Angelova, 2006).In vitro anticalicivirus efficacy of ribavirin was described by Povey (1978) who reported a marked effect on the FCV strain 255 by partial to complete suppression of the viral CPE in cell cultures.Antiviral activity of ribavirin was confirmed also by our previous study with FCV strain F9 (Tumbarski and Galabov, 2008).
As seen from the results in Table 4, ribavirin revealed a significant inhibitory activity when applied in the medium at the first four hours p.i. Adding of ribavirin during the late lag phase of the replication led to a marked and prolonged decrease of the infectious titer of FCV-F9 with 2.5 log CCID 50 /mL at 4 th h p.i. Application of ribavirin in exponential phase did not reduce the FCV-F9 titer and it reached the peak level at 8 th h p.i. (compared to the relevant controls).

Antiviral activity and kinetics of the inhibitory effect of PTU-23 on the replication of FCV-F9
The antiviral compound PTU-23 showed a low cell protective effect against FCV-F9 at virus inoculation doses 100 CCID 50 and 10 CCID 50 and low selectivity index -5.59 and 8.26 respectively (Table 2).PTU-23 is an antiviral compound that inhibits the synthesis of viral RNA, as a result of suppression of the synthesis of a viral protein with a regulatory function in the replication cycle (Galabov and Angelova, 2006).PTU-23 demonstrated antiviral effect when added to the maintenance medium during the lag phase of the FCV-F9 replication.This led to a reduction of the infectious virus titer with 2 log CCID 50 /mL at 3 rd h p.i. compared to the virus control levels.PTU-23 did not affect the replication when added in the exponential phase and FCV-F9 rapidly reached the peak titre at 8 th h p.i. (Table 5).

Table 2 :
50% cytotoxic concentrations (CC 50 ) and antiviral activity of the tested compounds against FCV-F9 expressed as 50% inhibitory concentration (IC 50 ) and selectivity index -SI (CC 50 /IC 50 ) by neutral red uptake assay.CC 50 and each IC 50 were calculated as mean value of two experiments/replicates. * Antiviral effect of ribavirin on the replication of FCV-F9 in one-step virus growth cycle.
*The results were calculated as mean value of two experiments/replicates.**KV -virus control.Tablе 4: *The results were calculated as mean value of two experiments/replicates.**KV -virus control.

Table 5 :
Antiviral effect of PTU-23 on the replication of FCV-F9 in one-step virus growth cycle.
*The results were calculated as mean value of two experiments/replicates.**KV -virus control.