Antiviral efficacy of favipiravir against canine distemper virus infection in vitro

Background Canine distemper (CD) is an acute infectious disease with high morbidity rates caused by a highly contagious pathogen (Canine Morbillivirus, also known as canine distemper virus, CDV). CDV can infect a broad range of carnivores resulting in complex clinical signs. Currently, there is no effective method to treat for CDV infections. Favipiravir (T-705), a pyrazine derivative, was shown to be an effective antiviral drug against RNA viruses, acting on RNA-dependent RNA polymerase (RdRp). However, whether the T-705 has antiviral effects following CDV infection is unclear. Here, we investigated the antiviral effect of T-705 against CDV-3 and CDV-11 strains in Vero and DH82 cell lines. Results Our data demonstrated that T-705 significantly inhibited the replication of CDV-3 and CDV-11 in both Vero and DH82 cells at different concentrations, ranging from 2.441 μg/ml to 1250 μg/ml. Additionally, T-705 exhibited efficacious antiviral effects when administered at different time points after virus infection. Cytotoxicity tests showed a slight decline in viability in Vero cells after T-705 treatment, and no apparent cytotoxicity was detected in T-705 treated DH82 cells. Comparison of anti-CDV polyclonal serum only inhibition of CDV in supernatant, T-705 directly inhibited viral replication in cells, and indirectly reduced the amount of virions in supernatant. The combination application of T-705 and anti-CDV polyclonal serum exhibited a rapid and robust inhibition against virions in supernatant and virus replication in cells. Conclusions Our data strongly indicated that T-705 effectively inhibited viral replication following CDV infection in vitro, and could be a potential candidate for treatment for CD.

Currently, no antiviral drug has been approved for therapeutic application in wildlife animals against CDV infection. The routine vaccination against CDV has been widely conducted for many years. Modified live vaccines (MLV) have significantly reduced CDV infections in dogs and other carnivores [16]. However, MLV are not completely safe in highly susceptible species [17], and CD outbreaks are known to occur even in vaccinated animals [8].
The genome of CDV is a single-stranded negativesense RNA, which codes a RdRP protein with a binding domain of ATP and/or purine ribonucleotide triphosphate [27,28]. Based on the efficacy of T-705 against RNA viruses in previous studies, our presumption is that T-705 may also work on CDV. Thus, in this study, we investigated the inhibitory effect of T-705 against two different CDV strains in Vero and DH82 cells, and compared the inhibitory effects of T-705 with an anti-CDV polyclonal serum. Our findings indicated that T-705 could be a potential anti-CDV drug.

Results
Growth characteristics of CDV-3 and CDV-11 in Vero and DH82 cells The growth characteristics of CDV-3 and CDV-11 strains in Vero and DH82 cells were determined by indirect immunofluorescence assay (IFA). Both Vero and DH82 cell lines inoculated with CDV-3 or CDV-11, exhibited a strongly positive reaction signal with anti-CDV N monoclonal antibody ( Fig. 1c-f); in contrast, no positive reaction signal with anti-CDV N monoclonal antibody was observed in mock cells ( Fig. 1a and b. In addition, 50% tissue culture infectious dose per milliliter assay (TCID 50 ) was used to determine the viral titers of cultured viruses at different time points (Fig. 1g). In Vero cells, viral titers of CDV-3 and CDV-11 peaked at 10 5.5 and 10 6.6 TCID 50 /ml at 72 h, respectively, and maintained a plateau between 72 h and 96 h. In DH82 cells, CDV exhibited a continuous increase of viral titers in the tested range of time points, and viral titers of CDV-3 and CDV-11 both peaked approximately at 10 5.5 TCID 50 /ml at 96 h.

Cytotoxicity of T-705
The CCK-8 method was performed on Vero and DH82 cells to determine the cytotoxic levels of T-705 (Fig. 2). A slight decline of viability of Vero cells treated with T-705 was observed at concentrations of 625 μg/ml and 1250 μg/ml. No obvious cytotoxicity was observed in T-705 treated DH82 cells for all tested drug concentrations. Moreover, the CC 50 (50% cytotoxic concentration) values of T-705 in both cell lines were estimated to be higher than 1250 μg/ml.

Inhibitory effects of T-705 on replication of CDV in Vero and DH82 cell lines
To investigate whether T-705 could inhibit replication of CDV, the antiviral effect of T-705 against CDV-3 and CDV-11 strains was evaluated in both Vero and DH82 cells (Fig. 3). The T-705 was simultaneously added with CDV strains to both cell lines. T-705 significantly reduced viral titers in both cell lines by a dose-dependent manner. Complete suppression of viral replication by approximately 5 or 6 logs was observed with T-705 at concentration levels of 78.125 μg/ml in CDV-11-infected Vero cells, 156.25 μg/ml in CDV-11-infected DH82 cells, and CDV-3-infected Vero and DH82 cells.

T-705 moment-of-addition experiment
Moment-of-addition experiments were performed to determine whether T-705 had effective and stable antiviral capacity in CDV-infected Vero and DH82 cells at different moments of drug additions. Inhibition percentages of T-705 were not as 100% as added immediately at 0 h, but still more than 30% in late moment of additions at 12 h to 48 h p.i.. In comparison of the mock-treated but virus-infected cells, viral titers were significantly reduced by 1000 times, when T-705 was added at 12 h to 48 h after viral infection (Fig. 4). Therefore, the T-705 has a strong inhibitory effect on CDV replication when added at 12, 24, 36 or 48 h p.i.. However, in this case, at the same moments of addition of the T-705, different concentrations did not significantly affect the virus titers, and the antiviral activity was similar in all tested concentrations of drug ( Fig. 4 e-h). These data indicated that even late intervention of CDV-infected cells with small concentrations of T-705 is still significantly effective.

Discussion
The routine vaccination against CDV MLV has been widely conducted for many years. However, MLV might cause severe diseases and deaths in highly susceptible species [17]. There were many cases of canine distemper outbreaks in vaccinated animals [8,29]. Cases of dogs suffering encephalitis after vaccination [29] have been reported in young dogs, and the immunity failure is mainly caused by inadequate immune responses to MLV with the presence of maternal antibody in puppy [30,31]. In addition, whether the MLV is suitable to the endangered species, needs to be considered for the variants with different susceptibilities among wild animals [32]. To address this problem, researchers investigated many antiviral drugs against CDV in vitro. Carvalho et al assessed the antiviral activity of several flavonoids and phenolic acids added at different time points [33]. Lanave et al assessed the antiviral efficacy of ribavirin and boceprevir alone or in combination against CDV [34]. Other studies showed that caffeic acid has significant anti-CDV effect in Vero cells [35]. María de Jesús González-Búrquez et al demonstrated the antiviral activity of the ethanolic extract of Mexican propolis, as well as the synergistic effect that exists between both studied favonoids [36]. Additionally, Krumm et al showed that treatment of CDV-infected ferrets, with an inhibitor which targeted the viral polymerase, could reduce viremia and prolong survival [37]. Other therapeutics including neutralizing monoclonal antibodies against the CDV hemagglutinin protein and nucleocapsid protein [38,39] showed potential immunotherapy against CDV infection in dogs. However, the polyclonal serum has disadvantages of limited sources and heterologous reactions, which restricted its clinical application, especially in wildlife and the endangered species. There is no effective antiviral molecular currently available to treat for CDV infection in susceptive animals. Thus, T-705, as a confirmed property, inhibits replication of a broad range of RNA viruses both in vitro and in vivo. Here, we have extended the spectrum of T-705, demonstrating robust antiviral activity against CDV in vitro. Our results in this work indicated that T-705 effectively suppressed CDV-3 and CDV-11 in Vero and DH82 cells. In Vero cells, the IC 50 of T-705 against CDV was 25.2 μg/ml for CDV-3 and 7.05 μg/ml for CDV-11. In DH82 cells, the IC 50 of T-705 against CDV was 33.54 μg/ml for CDV-3 and 16.97 μg/ml for CDV-11. These findings were comparable to those reported for other RNA viruses, including West Nile virus (IC 50 : 53 μg/ml) [40], foot-and-mouth disease virus (IC 50 : 14 μg/ml) [41], Ebolavirus (IC 50 : 10.5 μg/ml) [24], murine norovirus (IC 50 : 39 μg/ml) [25] and Zika virus (IC 50 :17.4 μg/ml) [42]. D. Jochmans et al demonstrated that recombinant MeV-Edm-GFP was the most sensitive to T-705 treatment of all tested paramyxoviruses, with 90% effective concentration (EC 90 ) values of 8.6 μM (1.7 μg/ml), 9.7 μM (1.9 μg/ml), and . Untreated cells were served as controls. Percentages of inhibition were measured by comparing the viral titer of drug-treated to control cells. All experiments were carried out in triplicate. Statistical significance was analyzed by one-way ANOVA (Dunnett's t-test). *, P < 0.01; **, P < 0.0001. ND means non-detectable 13 μM (2.56 μg/ml) for treatment prior to, simultaneous with, and after inoculation [26]. During CDV infection, viral particles are likely to directly attack the peripheral blood mononuclear, macrophages and other immune cells in hosts, and then transmit to bronchial lymph nodes and tonsils within the first 24 h p.i. [43][44][45]. Finally, it induces a long-term immunosuppression. In this study, T-705 showed robust antiviral effects against CDV in DH82 cells, a cell line from canine macrophages, which suggested that T-705 may be promising as a post-exposure therapeutic drug for CDV infection.
The moment-of-addition experiments proved that T-705 had significant antiviral effects against both CDV strains with addition between 0 and 48 h after virus infection in both cell lines. In previous studies, T-705 was shown to be phosphoribosylated to T-705RTP in cells by intracellular enzymes and then recognized as a purine analog by viral RdRp to inhibit viral replication [26,46]. That explained why a period of time was needed to reach an effective drug concentration. Virus titers of CDV exhibited a stable decline in DH82 and Vero cells, reaching roughly 10 3.5 or 10 4 TCID 50 /ml at 48 h after were determined and compared with the viral titers in mock group. Viral replications in cells were detected by IFA and the mean fluorescence percentages (a, c, e, g) were calculated as the average of fluorescent areas under twenty randomly selected fields. All experiments were carried out in triplicate. Statistical significance was analyzed by one-way ANOVA (Dunnett's t-test). *, P < 0.01; **, P < 0.0001 addition of T-705, in both cell lines, respectively. T-705 still exhibited a more than 30% inhibition rate on replications of CDV-3 or CDV-11 in Vero and DH82 cells even when added at 48 h after virus infection. The antiviral effects of T-705 against CDV showed concentration dependence and negative correlation with additional time. Previous studies demonstrated that the intervention effect of T-705 appeared at an early and intermediate stage of virus replication. In influenza virus, T-705 showed no effect when added at 6 to 10 h after infection [47]. In Tacaribe virus and Junín virus, there was little or no antiviral effect when T-705 was added at 18 h after virus infection [19]. Therefore, it is suspected that T-705 is more effective in combatting CDV infections as a post-exposure treatment. T-705 must have enough time to enter cells and switch to active form, before its antiviral effects exhibit. Comparisons of inhibitory effects between T-705 and anti-CDV polyclonal serum, showed that anti-CDV polyclonal serum only inhibited CDV in supernatant, while T-705 had little hinderance against CDV in supernatant directly. However, T-705 indirectly lessened virions concentration in supernatant by inhibiting viral replication in cells. T-705 combined with anti-CDV polyclonal serum showed an excellent and significant inhibitory effect in both supernatant and cells. Practical applicability of anti-CDV polyclonal serum was restricted by species specificity, but T-705 has a broad spectrum as an antiviral drug. The antiviral effect of T-705 against CDV in vivo was partially restricted for different susceptibility and pathogenicity of CDV in different animal models [48]. Our in vitro results indicated that T-705 was promising as a post-exposure treatment, and the antiviral effect of T-705 in vivo will be evaluated in a future study.

Conclusions
In this study, our findings demonstrated that T-705 could effectively suppress viral replication in CDV infected Vero and DH82 cells, indicating strong potential of T-705 as a treatment of CDV infections in the future. FBS supplied DMEM, and titrated by 50% endpoint titration. Anti-CDV polyclonal serum was purchased from Changchun Sino Biological Technology Co. Ltd.,

Cell viability
Viability of Vero and DH82 cells was performed using the Cell Counting Kit-8 (CCK-8, Dojindo, Japan). Briefly, Vero or DH82 cells at 80-90% confluence were grown in a medium containing different concentrations of T-705 in 96-well plates. After incubation at 37°C for 96 h, 10 μl of CCK-8 solution was added and incubated for a further 2 h at 37°C. The absorbance at 450 nm was determined. The cell viability was measured by comparing T-705-treated and mock-treated cells. This experiment was carried out in triplication.

Virus titration
The viral titers of both strains were determined by 50% endpoint titration in Vero cells and expressed as TCID 50 /ml. Vero cells in 96-well plates were combined with serial 10-fold dilutions of the virus, and incubated for 72 h at 37°C. Cells were fixed with 80% ice-cold acetone. Virus concentrations were measured by IFA. Cells were stained with CDV monoclonal antibody specific to nucleoprotein (VMRD, USA) and Alexa Fluor 488-conjugated Affinipure Goat Anti-Mouse IgG(H + L) (Proteintech, China). The cells were examined by fluorescent microscopy. Mock treated cells were used as a negative control. This experiment was carried out in triplication.

Viral inhibition assay
To determine the effect of T-705 against CDV in vitro, the culture medium of Vero or DH82 cells infected with CDV-3 or CDV-11 at a MOI of 0.  .)) were set to investigate whether T-705 could still show effective and stable antiviral activity during late administration of drug. In both cases, at 96 h p.i., cells and the culture medium were collected by freezing and thawing three times, and viral titers were measured by IFA as described previously. This experiment was carried out in triplication.

Comparison of antiviral effects of T-705 and anti-CDV polyclonal serum
The titer of virus neutralizing antibody (VNA) of anti-CDV polyclonal serum was 1:1024. A 250 μl of anti-CDV polyclonal serum was diluted in a 2 ml of DMEM, and the mixture has a final VNA titer of 1:128. The diluted polyclonal sera were tested for inhibition effects in a 6-well plate.
To evaluate the inhibitory effects of T-705 and anti-CDV polyclonal serum, the CDV-3 or CDV-11-infected Vero and DH82 cells were added with T-705 (156.25 μg/ml), anti-CDV polyclonal serum (1:128) and a combination of T-705 (156.25 μg/ml) and anti-CDV polyclonal serum (1:128) at 0, 12, 24, 36 and 48 h, respectively. The mock group was treated with 2% FBS DMEM after infection with CDV-3 or CDV-11. Viral titers in supernatant and viral replication of CDV in cells were determined by IFA at 96 h p.i.. Antiviral effects of tested groups were compared with mock groups treated with 2% FBS DMEM of CDV-infected cells. This experiment was carried out in triplication.

Statistical analysis
The data were displayed as the mean ± standard deviation (SD). Differences among viral titers were analyzed using GraphPad Prism 6 by one-way ANOVA (Dunnett's t-test). Statistical significance was considered at * P < 0.01 and ** P < 0.0001.