Skip to main content
NCBI home page
VirusDisease logoLink to VirusDisease
. 2021 Oct 20;32(4):737–747. doi: 10.1007/s13337-021-00746-8

Ribavirin as a curative and prophylactic agent against foot and mouth disease virus infection in C57BL/6 suckling and adult mice model

Patel Nikunjkumar 1, Ramasamy Periyasamy Tamil Selvan 1, Veerakyathappa Bhanuprakash 1,✉
PMCID: PMC8630122  PMID: 34901324

Abstract

Despite the availability of control measures for foot-and-mouth disease (FMD), the application of antiviral agents is imperative due to certain limitations in the prevention and control of FMD. This study pertains to systematic in vivo investigation of ribavirin as a prophylactic/curative agent, both in suckling and adult C57BL/6 mice against foot-and-mouth disease virus (FMDV) infection. In the adult mice, antiviral efficacy was assessed based on standard clinical score, body weight, and viral load. Only 13.33 to 33.33% of adult mice exhibited disease-specific symptoms following treatment and infection and vice versa, respectively, indicating the antiviral efficacy of the ribavirin. Further, the distribution of virus in different vital organs following ribavirin treatment and virus infection, and vice versa using SYBR green-based real-time PCR is reported. In the blood sample, the viral RNA was detected as early as two days post-infection and there was a significant reduction in virus titer (1000 to 10,000-folds) in the treatment groups compared to the infection control group. Animals receiving ribavirin had significantly lower organ virus titers at 2, 4, 6, 9, and 14 days post-challenge (dpc) than placebo-treated. In suckling mice, the treatment groups were 100% protected/cured compared to the control group. Thus, our data demonstrate that ribavirin may provide a feasible therapeutic approach to prevent as well as to treat FMDV infection.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13337-021-00746-8.

Keywords: Antiviral, C57BL/6 mice, Foot-and-mouth disease virus, Mouse model, Ribavirin, Toxicity

Introduction

Foot-and-mouth disease (FMD) is an economically devastating disease, affects cloven-footed animals including livestock and wild animals [39]. The disease is caused by the foot-and-mouth disease virus (FMDV), belonging to the genus Aphthovirus of the Picornaviridae family. The virus has seven antigenically distinct serotypes including O, A, C, Asia1, SAT1, SAT2, and SAT3. Each serotype includes various subtypes [4]. Presently, there is no treatment available for the disease. However, palliative treatment may be provided to diseased animals. The disease control measures have some limitations. Stamping out policy is expensive and difficult if the disease is widespread. Further, vaccine-induced protective immunity develops 4–7 days post-vaccination and there is little or no cross-protection between the serotypes [29]. Therefore, several alternate strategies have been attempted across the globe to combat FMD with varying degrees of success [13, 27]. In this direction, antiviral therapy has evinced promising effects in controlling the FMD virus replication [10, 11]. The antiviral compounds can be used either as therapeutic or prophylactic agents to reduce the disease spread.

Ribavirin is an antiviral agent (synthetic purine nucleoside analog) with antiviral activity against a wide range of RNA and DNA viruses [12]. The antiviral activity of ribavirin is attributed to several mechanisms [14] and it is advocated in several infections [5, 22]. There are in vitro studies suggesting the effectiveness of ribavirin on different serotypes of FMDV replication [1, 10, 18, 32, 35, 38], but information on in vivo studies is scanty [3, 18]. Choi and co-authors have reported that the ribavirin had showed better antiviral activity in comparison to 6-azauridine or T1105 against FMDV O/SKR/2002 and synergistic antiviral activity when administered in combination with FMD vaccine in mice and pig [3]. To study the curative/prophylactic effect of an antiviral, selection of a best laboratory model for the study was imperative. Hence, thorough literature search yielded that the FMDV A serotype is lethal to suckling [15] and adult mice [19] compared to O and Asia 1. The susceptibility of adult mice depends on virus specific factors such as the virus strain, serotype, passage history; and host specific factors like mouse strain and the route of infection [19, 23]. Among the laboratory mice, C57BL/6 mice are the most susceptible strain to FMDV infection [15]. Therefore in the present investigation, pathogenesis of FMDV A as well as the inhibitory effect of ribavirin as preventive and therapeutic agent on FMDV A in suckling and adult mice models have been investigated.

2. Materials and methods

2.1. Antiviral compound

Ribavirin was purchased commercially (Sigma, MO, USA) and stored at 4 °C till use. The drug was reconstituted in sterile double distilled water and working aliquots of ribavirin were stored at −20 °C till use.

2.2. Virus and animals

Indian vaccine strain, FMDV serotype A (A/IND/40/2000) passaged (P4) in cattle was maintained at the FMD research center, ICAR-Indian Veterinary Research Institute (ICAR-IVRI), Bengaluru was used. C57BL/6 mice procured commercially were used throughout the experiment. The virus was passaged in suckling mice (2–4 day old) prior to use in the study. Animals were quarantined for 48 h prior to animal experiment and fed standard mouse feed and water ad libitum. All the animal experiments were performed according to institutional and national ethical guidelines. Necessary approval of the Committee for the purpose of control and supervision of experiments on animals (CPCSEA) and Institute biosafety committee (IBSC) was obtained prior to animal experiments.

In vivo passage and titration of FMDV serotype A

The FMDV A/IND/40/2000 was passaged three times in 2–4 day old suckling mice. In brief, 50 µl of virus suspension in PBS inoculated intramuscularly in the left hind limb and mice were observed for clinical signs till death. After death, the skeletal muscles were collected and triturated in sterile PBS (pH 7.4) with a ratio of 1:10 (W/V) [34]. The triturated material was centrifuged at 3500 rpm for 10 min at 4 °C and supernatant was filtered using a 0.22 μm syringe filter (Merck, Darmstadt, Germany). Fifty microliter of filtrate was used to infect fresh and healthy suckling mice in the subsequent passage. Likewise, three successive passages were done and the virus suspension from the third passage was collected and termed as FMDV A [P3] virus and stored at −80 °C till use. The identity of the virus at each passage was confirmed by serotype specific sandwich ELISA and multiplex PCR (using NK61 and DHP15-serotype specific primer) [9]. Further, the FMDV A [P3] virus suspension was titrated in sucking mice (4–5 days old) and adult mice (6–7 week old) by intramuscular and intraperitoneal routes respectively. Five C57BL/6 sucking mice were used for each dilution of the virus and the death was considered as the end point. In case of adult mice, three mice were used per dilution and the development of clinical disease considered as the end point for virus titration. Virus titer was calculated as per Reed and Muench (1938) [26]. FMDV A [P3] was used for challenging the adult (6–7 week old) and suckling mice (5–6 days old), while assessing the in vivo antiviral activity of ribavirin.

Antiviral efficacy of ribavirin

In adult mice

The efficacy of ribavirin as therapeutic or prophylactic agent against FMDV A was assessed in adult mice. Four groups of mice included were ribavirin pre/post-treatment, infection and healthy control. Four groups of mice, each group with 15 (n = 15) mice were maintained except the healthy control group, where 13 (n = 13) mice were maintained. In case of pre-treatment group, each adult C57BL/6 mouse was administered with ribavirin at 50 mg/kg/day by intraperitoneal route twice daily for three days followed by challenging of each mouse with 100 ID50 (in 100 μl) of FMDV A [P3] virus intraperitoneally after 24 h post drug administration. However, in case of post-treatment group, each mouse was infected with 100 ID50(in 100 μl) of FMDV A [P3] virus intraperitoneally followed by treatment of each mouse with ribavirin at 50 mg/kg/day intraperitoneally after 24 h post infection twice daily for 3 days. In the case of the infection control group, each mouse was infected with 100 ID50 (in 100 μl) of FMDV A [P3] intraperitoneally. In the healthy control group, each mouse was neither treated with ribavirin nor infected with the virus. All the animals in each group were given feed and water ad libitum. The clinical signs and symptoms such as decreased appetite, decreased body weight, arch-back posture, tucked-up abdomen, paresis, paralysis and death, if any, were observed for 15 dpc.

2.4.2. Sacrifice and sampling

Three mice from each group were sacrificed on 2, 4, 6, 9 and 14 days post challenge. The blood samples and organs were collected. The vital organs like lung, liver, pancreas, muscle, heart, thymus, spleen and kidney were harvested from each mouse, weighed and preserved in 50% phosphate glycerol saline at−80 °C until use. Later, the pool of each organ was thawed and homogenized in PBS (pH 7.4) using sterile mortars and pestles. Virus from each organ pool was quantified in real time PCR as per gram tissue. Similarly, the virus titre in blood was quantified using real time PCR and expressed per 100 μl of blood.

2.4.3. In suckling mice

The efficacy of ribavirin as a curative or prophylactic agent against FMDV A was assessed in 5- 6 day old C57BL/6 suckling mice. The mice were divided into four groups, each group with five mice. The four groups included ribavirin pre/post-treatment, infection control and healthy mice. In case of pre-treatment group, each suckling mouse was administered once with ribavirin at 50 mg/kg by intraperitoneal route followed by challenge with 100 suckling mouse lethal dose (SMLD50) of C57BL/6 mouse adapted virulent FMDV A in 100 μl by intramuscular route after 12 h post drug administration. In the case of post-treatment group, each mouse was infected with 100 SMLD50 of FMDV A [P3] (in 100 μl) intramuscularly followed by administration of ribavirin at 50 mg/kg intraperitoneally after 6 h post infection. Whereas, in the case of infection control group, each mouse was infected with 100 SMLD50 of FMDV A [P3] in 100 μl intramuscularly. In the case of the healthy control group, all the mice were neither treated with ribavirin nor infected with FMDV A [P3] but administered with 100 μl of sterile PBS intramuscularly. The clinical signs of disease and death were observed for seven days.

Evaluation of drug toxicity

The toxicity of ribavirin at its treatment dose was determined both in adults as well as suckling C57BL/6 mice. In the case of adult mice, two groups (drug control group and normal mice control group) with 15 (n = 15) mice in each group were maintained. In the drug control group, each adult mouse was administered with ribavirin at a treatment dose of 50 mg/kg/day twice daily (in 100 μl) for three days intraperitoneally. Each control mouse in the normal mice control group was administered with 100 μl of sterile PBS intraperitoneally. The ribavirin toxicity at treatment dose was evaluated based on body weight and total RBC count. The animal body weights were determined prior to the first treatment up to 15 days. Blood samples from three animals (randomly) from each drug control and normal mice control group were collected daily (facial vein) in a tube containing EDTA for total RBC count. Total RBC counts were done manually using a haemocytometer.

In case of suckling mice, two groups viz., drug control and normal mice control groups were maintained. In each group, 5 (n = 5) suckling mice were utilized. In the drug control group, each suckling mice was treated once with ribavirin at a dose of 50 mg/kg body weight intraperitoneally (in 100 μl). Ribavirin toxicity in suckling mice was evaluated by weight loss, if any, which is a sign of ribavirin induced reversible anaemia [16]. The animal body weights were determined daily for 7 days. All the parameters were compared with normal mice control mice group.

Quantification of FMDV nucleic acid from timed samples by SYBR green based Quantitative PCR

Total RNA from different timed samples was isolated using the TRIzol reagent (Invitrogen, CA, USA) as per the instructions of the manufacturer. The concentration of the extracted RNA was measured using a NanoDrop (BioTek, VT, USA). Real time-PCR was performed using the SYBR Green I master mix, a two-step RT-PCR Maxima SYBR Green kit (ThermoFisher Scientific, MA, USA). Real time PCR was performed in 7500 ABI real time PCR machine (Applied Biosystems, CA, USA). Reaction was conducted in 20 μl using 10 μl of SYBR Green PCR 2x-Master mix, 2 μl of cDNA template, 0.6 μl (10 pmol/μl) each of gene specific forward (DHP-15) and reverse primers (NK-61) and the reaction volume was adjusted to 20 μl with nuclease free water. Real-time PCR was done with initial denaturation at 95 °C for 10 min followed by 50 cycles of amplification with denaturation at 95 °C for 15 s, annealing at 60 °C and extension at 72 °C for 30 s for each cycle. Specificity of the amplified product was assessed by dissociation curve, generated at temperature 55 °C through 95 °C. The result was expressed as cycle threshold values (Ct). Ct value is the cycle number when the fluorescence of the reporter dye (SYRB Green) is appreciably higher than the background fluorescence. Quantity of viral RNA was estimated as viral RNA log10TCID50 equivalent from known standard dilution series of FMDV A. The standard dilution series of FMDV A was used to construct standard curves from which the quantity of viral RNA (as viral RNA TCID50 equivalent) of each timed sample of serotype A obtained from respective drug treatment processes were estimated. The threshold automatically adjusted by the instrument is used for the generation of Ct values.

Statistical analysis

The difference in body weight was compared by two-way ANOVA using GraphPad Prism version 5.0. The disease scores of mice of different groups were compared day-wise with the non-parametric Kruskal–Wallis Test. Dunn's multiple comparison tests were used to compare the infection group with treatment groups. Analysis of virus titer in blood was carried out using Kruskal–Wallis Test with Dunn's multiple comparison tests. Kaplan–Meier survival curve analysis was done for the time taken to death after infection and treatment groups. **P- value of < 0.05 significant and ***P-value of < 0.001 or < 0.0001 considered highly significant.

Results

In vivo virus passage and disease characteristics of FMDV A infection in mice

Initially, the FMDV A was passaged in suckling C57BL/6 mice followed by induction of disease in suckling and adult mice were studied. For passaging the FMDV A in mice, 10% bovine tongue epithelial suspension in 50 μl of FMDV A (105.20Bovine Infective Dose50/ml) was administered to 2–4 day old suckling mice intramuscularly in the left hind limb. The initial mortality in suckling mice was noticed at 16 hpc without any clinical signs of the disease. The remaining mice had paralysis of virus inoculated limb. Further, the disease progression leads to paralysis of the other hind limbs followed by forelimbs and finally the death of the mice (Fig. 1a). While passaging the virus, progressive flaccid paralysis of all four limbs (quadriplegia) followed by death was observed in all the cases. The flaccid paralysis was manifested by disappearance of normal splaying of the toe of affected limb as reported earlier [34]. As the virus has been known to concentrate in heart muscles and skeletal muscles, skeletal muscle tissues were collected from dead mice for subsequent passaging of virus. The signs of disease exhibited by the suckling mice upon passaging with FMDV A were alike, irrespective of the passage number of the virus. Virus suspension from third passage (FMDV A [P3]) was titrated in mice and used as a challenge virus for antiviral study. In suckling mice, FMDV A [P3] infection showed similar signs as shown during virus passaging. The virus titre in suckling mice was 107.67 SMLD50/ml.

Fig. 1.

Fig. 1

Open in a new tab

a 4–5 day old suckling mice showing progressive paralysis followed by death, when infected with FMDV A [P3] virus by intramuscular route. 1, Normal mice; 2, Paralysis in right hind limb at 16 hpc (hour post challenge); 3, Paralysis of both hind limbs at 26 hpc; 4, Paralysis in both hind limbs and a forelimb at 32 hpc; 5, Paralysis of all limbs (quadriplegia) at 35 hpc; 6, Death at 36 hpc. b Clinical sings of disease observed in adult mice. Six to seven weeks old mice were intraperitoneally inoculated with 100 MID50 of FMDV A [P3]. Disease scoring: 0, Normal mice; 1, Apathic mice; 2, Humped posture/ Arch-back condition with ruffled fur; 3, Tucked up abdomen with wasting condition; 4, Mild hind limb paralysis

In adult mice, virus induced death was rare but a characteristic disease pattern was observed. Following inoculation, the first sign of the disease was apathy with increased respiration, observed after 24 hpc. At 48 hpc, the mice were apathic and dull with ruffled body coat and after 72 hpc, there was a wasting condition with reduced body weight. Mice had humped posture (arch-back) condition followed by tucked-up abdomen with increased abdominal respiration and change in gait. On 4 to 5 dpc, mice exhibited neurological signs like mild paralysis of limbs, especially the hind limbs. The signs of the disease were predominant between 2 and 6 dpc. Animals did not show any vesicular lesions around the foot pad that are characteristic of a natural host.On the 5th dpc, there was a reduction in the body weight up to 30%, compared to the initial body weight. Animals started showing recovery after 10 dpc. Based on the clinical disease observed, the mouse infective dose of FMDV A [P3] was 104.4 mouse infective dose 50 (MID50)/ml. The clinical signs noticed are shown in Fig. 1b, which were further used to evaluate the antiviral efficacy of ribavirin.

3.2. Antiviral efficacy of ribavirin in adult mice model

3.2.1. Inhibition of clinical disease

Suckling mice passaged FMDV A [P3] did not induce death in adult mice. Therefore in this model, the antiviral efficacy of ribavirin was evaluated based on inhibition of clinical disease (disease scoring system) and inhibition of viral RNA load in blood and various organs/tissues. Infection of mice with 100 MID50 resulted in the development of the disease in all the mice in the infection control group. In pre and post-treatment groups the disease observed was only in 13.33% and 33.33% of mice, respectively (Fig. 2). As far as the body weight is concerned, on 7thdpc, an average < 1%, < 8% and ~ 20% reduction in the body weight was observed respectively in pre-treatment, post-treatment and infection control groups compared to healthy control group (*p < 0.05). At the end of the study (14 dpc), the average body weight of mice in infection control group was ~ 12% less, whereas, no significant difference was observed in both treatment groups, compared to the healthy control group (*p > 0.05) (Fig. 3).

Fig. 2.

Fig. 2

Open in a new tab

Treatment of adult mice with ribavirin at 50 mg/kg/day twice daily for three days inhibited the signs of disease in 100 MLD50 challenged adult mice. Both treatment groups (n = 15) showed significant reduction in disease score compared to infection control groups (n = 15). A highly significant difference (**P < 0.001) in the median disease score was observed between groups starting from day 2 onwards until day 7. The difference in rank-sum was more between infection group and the pre-treatment group compared to the post-treatment group

Fig. 3.

Fig. 3

Open in a new tab

Treatment of adult mice with ribavirin at 50 mg/kg/day twice daily for three days prevented or reduced the loss of body weight in pre-treatment (n = 15) and post-treatment groups (n = 15) compared to healthy control group (n = 15). Whereas, in case of infection group (n = 15), there was a significant loss of body weight of mice compared to the healthy control group (***P < 0.0001)

Inhibition of virus titre in blood and other organs

Blood and organ samples collected from various days post challenge from all the groups had been subjected to SYBR green based real time PCR. Viral RNA in blood was detected only on 2 dpc. At 2 dpc, the reduction in the virus titre in the blood was 1000- and 10,000-folds in pre and post treated mice groups, respectively (*p > 0.05) compared to the infection control group (Fig. 4). There was a significant reduction in virus titre in the organs collected in ribavirin treated groups compared to the infection control mice (Table 1).

Fig. 4.

Fig. 4

Open in a new tab

Treatment of adult mice with ribavirin at 50 mg/kg/day twice daily for three days inhibited blood virus titre on 2 dpc. Comparison of virus level (Log10SMLD50 equivalent) in pre and post-treatment groups at 2 dpc showed that there is significant difference in median titer among them (*P < 0.05)

Table 1.

FMDV viral RNA copy numbers detected by Q-PCR from various organ pools at different days post infection (dpi) from infection, pre and post treatment groups

Organs Placebo group (Days post infection) Pre-treatment group (Days post infection) Post-treatment group (Days post infection)
2 4 6 9 14 2 4 6 9 14 2 4 6 9 14
Thymus 5.3 4.56 – – – – – – – – – 2.1 – – –
Liver 3.1 2.9 – – – – 0.1 – – – 2.3 0.6 – – –
Pancreas 5.5 4.4 2.1 2.1 – 3.7 1.2 0.2 1.3 – 7.3 0.5 0.2 1.1 –
Lungs 4.6 4.3 3.1 2.7 – 4.1 3.1 2.5 2.6 – 5.0 3.2 2.9 3.27 –
Heart 6.0 4.8 3.0 2.7 – 4.0 3.3 3.0 – – 5.6 2.7 3.2 – –
Kidney 4.5 4.4 3.2 3.9 – 4.6 3.9 3.0 3.5 – 5.5 3.7 3.0 3.6 –
Spleen 6.6 5.8 3.9 3.26 4.2 6.8 4.5 3.51 2.9 3.3 7.2 5.0 3.6 2.8 4.6
Muscle 3.3 3.2 0.9 1.3 – 2.7 1.5 – – – 3.8 1.3 0.8 1.9 –
Open in a new tab

Virus titer of three organ pools (log10 SMLD50)

Antiviral efficacy of ribavirin in suckling mice model

The antiviral efficacy of ribavirin was evaluated against lethal infection caused by FMDV A [P3]in suckling mice. Four groups of mice used were pre and post-treatment, infection and healthy control groups. Mice were infected with 100 SMLD50 of FMDV A [P3] intramuscularly. In the pre and post-treatment groups, treatment was given at 12 h before and 6 h after virus challenge, respectively, as the virus causes disease or death within 16 hpc. There was no clinical sign like paralysis of limb or any mortality was observed in the pre-treatment group throughout the study period. In the post-treatment group, only one animal showed a sign of respiratory distress and mild weakness in virus inoculated limb but it recovered within 48 h after the onset of the signs. In case of infection control group, all the animals died within 48 hpc with the first casualty at 28 hpc. All the dead mice showed clinical signs of the disease prior to their death that was similar as described above. The survival rate in case of both treatment groups was 100% (Fig. 5).

Fig. 5.

Fig. 5

Open in a new tab

Antiviral efficacy of ribavirin in 7 day old C57BL/6 suckling mice. Infection group (n = 5), pre-treatment group (n = 5) and post-treatment group (n = 5) mice were challenged with 100 SMLD50 of mice passage 3 FMDV A virus (50 µl intramuscularly in left hind limb). In pre-treatment group (n = 5) suckling mice were treated once with ribavirin at 50 mg/kg body weight intraperitoneally, 12 h before virus challenge. In post-treatment group mice were treated once with ribavirin at 50 mg/kg body weight intraperitoneally, 6 h after virus challenge. In infection control group placebo treatment was given. In heath control group (n = 5) mock treatment/infection was given. Survival rate was monitored for 7 days post virus challenge (*P < 0.05)

3.4. Determination of ribavirin toxicity

Ribavirin toxicity at its treatment dose for mice was determined based on the total RBC count and the loss in body weight. It was found that the ribavirin at a dose of 50 mg/kg, twice daily for three days intraperitoneally, did not induce any hematotoxicity (***p < 0.0001) (Fig. 6a) or loss in the body weight of adult mice (*p < 0.05) (Fig. 6b). In case of suckling mice, it was observed that a single dose of ribavirin at 50 mg/kg body weight administered intraperitoneally did not cause significant loss of body weight. However, 48 h following drug administration, there was a mild reduction in average body weight compared to healthy control group, but at the end of study on 7th dpi, the mean body weight of drug control group was significantly similar as that of healthy control group, indicating the treatment dose of ribavirin was well tolerated by 5–6 day old suckling mice (*p < 0.05) (Fig. 6c).

Fig. 6.

Fig. 6

Open in a new tab

a Ribavirin at 50 mg/kg/day twice daily for 3 days is non-toxic to adult (6–7 weeks old) mice. Groups (n = 8) of C57BL/6 mice were treated placebo and ribavirin at 50 mg/kg/day twice daily for 3 consecutive days. Total RBC count in ribavirin administered mice showed a significant decrease (*P < 0.05) on day 7 compared to ribavirin denied mice. However, the RBC count on different days remained the same. b The animals were weighed before treatment and continued for 14 day. There is no significant difference (***P < 0.0001) in body weight between ribavirin administered and denied mice indicating the absence of ribavirin toxicity. c Single dose ribavirin at 50 mg/kg body weight administered intraperitoneally is not toxic to 5–7 day old C57BL/6 suckling mice. Normal mice control group (n = 5) were treated with placebo (PBS) and drug control group (n = 5) treated with single dose of ribavirin at 50 mg/kg body weight. The animals were weighed daily for 7 days. Values in graph represent the mean of mouse body weight of each group per day (***P < 0.0001)

4. Discussion

Several alternative strategies have been attempted across the globe to combat FMD with varying levels of success apart from current control measures including stamping out policy (mass culling) and vaccination with inactivated vaccine [11]. Some of the promising alternative strategies to combat FMDV include use of immunostimulants, RNA interference (RNAi), interferon therapy and chemical compounds either alone or in combination with vaccine has been described [11, 17]. Antiviral agents can be used as alternatives and/or supplementary molecules to control FMD. However, the effectiveness of antiviral agents depends on its efficacy, specificity, toxicity and drug-resistance profile [13]. Among the antiviral compounds, ribavirin is one of the promising molecules. Ribavirin exhibits antiviral activity directly by interfering with RNA capping, viral polymerase inhibition, lethal mutagenesis in viral genome and indirectly by inhibiting inosine monophosphate dehydrogenase (IMPDH) enzyme, which reduces the intracellular guanosine triphosphate concentration and affects viral protein synthesis. Inhibition of IMPDH alone may not be enough for antiviral activity of ribavirin. Moreover, FMDV replication is cap independent, without involvement of 2-O-methyltransferase and capping enzymes. Hence, the possible antiviral mechanism of ribavirin against FMDV was due to lethal mutagenesis and inhibition of viral RNA dependent RNA polymerase. Besides these, ribavirin is shown to modulate host immune response by enhancing T-cell response [3, 18]. C57BL/6 mice strain was used in the present experiments. Accordingly, FMDV A was passaged in suckling mice to study the FMDV infection and to evaluate antiviral efficacy of ribavirin against FMDV infection. Previously, severe combined immune deficient (SCID) mice strain was used to develop a model for FMDV infection and preliminary evaluation of antiviral drugs [19]. FMDV serotype A (A/IND/40/2000) was serially passaged in 2–4 day old C57BL/6 mice to increase its in vivo virulence and pathogenicity of the virus to mice as was done earlier [33, 36]. The most consistent clinical features observed in suckling mice were weakness and progressive flaccid paralysis of limbs followed by death within 72 hpc. Progressive paralysis of limbs is the result of virus replication in skeletal muscles [34]. Virus causes lethal disease in suckling mice but the level of lethality depends on serotype/strain of the virus [8, 15]. In the present study, the suckling mice died within 72 hpc indicating that FMDV serotype A (A/IND/40/2000) strain is highly virulent to suckling C57BL/6 mice strain. The paralysis of limbs was the first and predominant sign of the disease as observed. The progressive flaccid paralysis of limbs followed by death observed in FMDV A/40/2000 infection in suckling C57BL/6 mice strain is similar to the earlier report [24], indicating the commonality of the disease symptoms of FMDV in suckling mice irrespective of virus serotypes. Our findings support the myotropic nature of FMD virus in suckling mice which could be the direct cause of limb paralysis. Acute flaccid paralysis in young mice has also been reported with other picornaviruses (enterovirus 71 and Coxsackie A viruses) infection resulted due to extensive replication of the virus in skeletal muscle tissues [21, 42].

Though FMDV can cause lethal disease in suckling mice and can be used for evaluating the efficacy of antiviral compounds, adult mice are more preferable owing to their similarity of kinetics/pathogenesis of virus infection with the cattle and other natural hosts. Both in the natural host and in adult mice, FMD infection is characterized by short duration of viremia, replication of virus in pancreas, clearance of virus from blood coinciding with an increase in the serum neutralizing antibody titre [6, 19, 30]. Outcome of FMDV infection in adult mice is variedly depends on the virus serotype/strain, amount of virus used, strain of mice and route of inoculation [15]. However, with few exceptions, the ability of FMDV to cause death in adult mice is limited [34, 37]. Therefore, it was intended to study the course and outcome of suckling mice passaged FMDV A virus (FMDV A [P3]) infection in adult C57BL/6 mice, so that it can be used as a model with death as the final outcome or to establish a defined disease scoring system to classify the mice as suffered from the disease or not. In the present study, FMDV A [P3] was unable to cause mortality in adult mice despite occurrence of clinical disease for 9 to 10 day post challenge. The observed clinical signs were apathy/malaise; humped posture with ruffled fur, tucked up abdomen with wasting condition (significant loss of body weight) and mild paralysis of limbs which correspond to previous studies [19, 30, 31, 36].

Here, disease scoring system was adopted to assess the antiviral efficacy of ribavirin, considering the severity of symptoms and time course of appearance of symptoms. Both treatment regimens (24 h before and after virus challenge) had shown effectiveness of ribavirin as it prevented the clinical disease in both treatment groups (pre-treatment group 86.66% and post-treatment group 66.66% animals were not shown any clinical sign of disease). In addition, ribavirin reduced the course and severity of the disease and thereby it can be used to reduce the duration of infectiousness and reduce the progress of outbreak. In the infection control group, after short term viremia, virus was detected in thymus, liver, pancreas, heart, lungs, kidney, spleen and muscles indicating the systemic infection in adult mice. However, there was a reduction in viremia in ribavirin treatment groups, indicating the antiviral effect of ribavirin and thereby reducing the further dissemination of virus to various organs/tissues. This is evident by the reduction of the virus titre in the organs and reduced severity of clinical signs. It has already been shown that ribavirin treatment (100 mg/kg daily for 9 days) reduced viral load in brain tissues and subsequently prevented enterovirus 71 in causing limb paralysis and mortality in 2 weeks old ICR mice [20], indicating that the action of ribavirin is irrespective of virus tested against it. Recently, it is demonstrated that ribavirin (10 mg/day) synergistically acts with vaccines to prevent FMDV induced mortality in 7 weeks old C57BL/6 mice by completely inhibiting viremia at 4 day post infection [3]. Together with this observation, our data also proves that the viremia could be shortened by treatment with ribavirin and thereby by its utility in outbreak situations to reduce the quantity of virus excreted from the infected host. It has been reported that after intramuscular or intravenous administration, ribavirin distributes in various organs and tissues including brain in rodents [7]. Ribavirin induces hemolytic anemia due to physiological extravascular destruction of ribavirin accumulated erythrocytes by the reticuloendothelial system. Extensive accumulation of ribavirin in erythrocytes causes intra-erythrocyte oxidative stress which subsequently leads to membrane damage [28]. The data from drug toxicity evaluation suggested that the ribavirin at a dose of 50 mg/kg/twice daily for 3 days intraperitoneally neither caused hemotoxicity nor reduction in the body weight. Previous studies have also indicated that ribavirin hemotoxicity has been found at a higher dose of 100 mg/kg/day [16].

Further, the antiviral efficacy of ribavirin was determined against the lethal infection of FMDV A in suckling mice. The observation during the course of seven days study indicates that the single dose of ribavirin (50 mg/kg) given at 12 h before and 6 h after, protected the mice from lethal infection with 100 LD50 of FMDV A [P3] with a survival rate of 100% (P = 0.05). Previous study has reported that the treatment of ribavirin at ~ 2 mg/kg body weight could protect 30 to 40% and 6%, when 6-day old ICR mice were challenged with 125 LD50 and 250 LD50 of FMDV O/SKR/2002, respectively [18]. This implies that the antiviral efficacy of ribavirin depends on the dose of ribavirin, the serotype/strain of FMDV and the amount of challenge virus used. Moreover, it is demonstrated that ribavirin induced FMDV mutant showed reduced virulence in suckling mice [40, 41]. The drug toxicity study indicates that, single dose of ribavirin at 50 mg/kg body weight was well tolerated by 5–6 day old C57BL/6 mice. This observation is in conformity with the previous reports that the ribavirin up to 80 mg/kg body weight once daily for five days administered intraperitoneally was well tolerated by a day old ICR suckling mice without showing any mortality for a period of 15 days post drug administration [43].

In conclusion, ribavirin as a prophylactic and therapeutic agent effectively inhibited FMDV A induced disease in adult mice and prevented death in suckling mice. The anti-FMDV effect of ribavirin after establishing infection in mice was relatively less compared to pre-treatment strategy. Despite that, the study results indicate ribavirin could be used as a preventive or therapeutic agent that could be used to break the transmission cycle of the virus and reduce the impact of outbreak in natural settings by inhibiting the virus replication at the onset of outbreak in valuable farm animals and/or susceptible endangered wild animals especially, in enzootic counties, where the zoo animals are at threat [25]. Though ribavirin alone is effective, the combination of ribavirin with other antiviral agents may increase the effectiveness of antiviral agents as well as it might reduce the chances of development of drug resistance [2, 3]. Further, pharmacokinetic and pharmacodynamic studies in the natural host like cattle are warranted to standardize the effective dose of ribavirin for its evaluation against FMDV infection.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (MP4 4359 kb) (4.3MB, mp4)
Supplementary file2 (MP4 4711 kb) (4.6MB, mp4)
Supplementary file3 (MP4 4087 kb) (4MB, mp4)
Supplementary file4 (MP4 8271 kb) (8.1MB, mp4)
Supplementary file5 (MP4 3908 kb) (3.8MB, mp4)
Supplementary file6 (MP4 3414 kb) (3.3MB, mp4)
Supplementary file7 (MP4 4616 kb) (4.5MB, mp4)
Supplementary file8 (MP4 6577 kb) (6.4MB, mp4)
Supplementary file9 (MP4 4608 kb) (4.5MB, mp4)
Supplementary file10 (MP4 4649 kb) (4.5MB, mp4)

Acknowledgements

Authors are thankful to the Director and the Joint Director, ICAR-Indian Veterinary Research Institute (IVRI), the Indian Council of Agricultural Research (ICAR), New Delhi and staff of FMD Research Laboratory, IVRI, Hebbal, Bengaluru, Division of Virology, ICAR-IVRI, for providing the necessary help and facilities.

Declarations

Conflict of interest

There was no conflict of interest among the authors.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  • 1.Airaksinen A, Pariente N, Menéndez-Arias L, Domingo E. Curing of foot-and-mouth disease virus from persistently infected cells by ribavirin involves enhanced mutagenesis. Virology. 2003;311(2):339–349. doi: 10.1016/S0042-6822(03)00144-2. [DOI] [PubMed] [Google Scholar]
  • 2.Borrego B, Blanco E, Pulido MR, Mateos F, Lorenzo G, Cardillo S, Smitsaart E, Sobrino F, Sáiz M. Combined administration of synthetic RNA and a conventional vaccine improves immune responses and protection against foot-and-mouth disease virus in swine. Antivir Res. 2017;142:30–36. doi: 10.1016/j.antiviral.2017.03.009. [DOI] [PubMed] [Google Scholar]
  • 3.Choi JH, Jeong K, Kim SM, Ko MK, You SH, Lyoo YS, Kim B, Ku JM, Park JH. Synergistic effect of ribavirin and vaccine for protection during early infection stage of foot-and-mouth disease. J Vet Sci. 2018;19(6):788. doi: 10.4142/jvs.2018.19.6.788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Domingo E, Escarmıs C, Baranowski E, Ruiz-Jarabo CM, Carrillo E, Núñez JI, Sobrino F. Evolution of foot-and-mouth disease virus. Virus Res. 2003;91(1):47–63. doi: 10.1016/S0168-1702(02)00259-9. [DOI] [PubMed] [Google Scholar]
  • 5.Dusheiko G, Main J, Thomas H, Reichard O, Lee C, Dhillon A, Rassam S, Fryden A, Reesink H, Bassendine M, Norkrans G. Ribavirin treatment for patients with chronic hepatitis C: results of a placebo-controlled study. J Hepatol. 1996;25(5):591–598. doi: 10.1016/S0168-8278(96)80225-X. [DOI] [PubMed] [Google Scholar]
  • 6.Fernández FM, Borca MV, Sadir AM, Fondevila N, Mayo J, Schudel AA. Foot-and-mouth disease virus (FMDV) experimental infection: susceptibility and immune response of adult mice. Vet Microbiol. 1986;12(1):15–24. doi: 10.1016/0378-1135(86)90037-4. [DOI] [PubMed] [Google Scholar]
  • 7.Ferrara EA, Oishi JS, Wannemacher RW, Jr, Stephen EL. Plasma disappearance, urine excretion, and tissue distribution of ribavirin in rats and rhesus monkeys. Antimicrob Agents Chemother. 1981;19(6):1042–1049. doi: 10.1128/aac.19.6.1042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.García-Nuñez S, König G, Berinstein A, Carrillo E. Differences in the virulence of two strains of foot-and-mouth disease virus serotype A with the same spatiotemporal distribution. Virus Res. 2010;147(1):149–152. doi: 10.1016/j.virusres.2009.10.013. [DOI] [PubMed] [Google Scholar]
  • 9.Giridharan P, Hemadri D, Tosh C, Sanyal A, Bandyopadhyay SK. Development and evaluation of a multiplex PCR for differentiation of foot-and-mouth disease virus strains native to India. J Virol Methods. 2005;126(1–2):1–1. doi: 10.1016/j.jviromet.2005.01.015. [DOI] [PubMed] [Google Scholar]
  • 10.Goris N, De Palma A, Toussaint JF, Musch I, Neyts J, De Clercq K. 2′-C-methylcytidine as a potent and selective inhibitor of the replication of foot-and-mouth disease virus. Antivir Res. 2007;73(3):161–168. doi: 10.1016/j.antiviral.2006.09.007. [DOI] [PubMed] [Google Scholar]
  • 11.Goris N, Vandenbussche F, De Clercq K. Potential of antiviral therapy and prophylaxis for controlling RNA viral infections of livestock. Antivir Res. 2008;78(1):170–178. doi: 10.1016/j.antiviral.2007.10.003. [DOI] [PubMed] [Google Scholar]
  • 12.Graci JD, Cameron CE. Mechanisms of action of ribavirin against distinct viruses. Rev Med Virol. 2006;16(1):37–48. doi: 10.1002/rmv.483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Grubman MJ. Development of novel strategies to control foot-and-mouth disease: marker vaccines and antivirals. Biologicals. 2005;33(4):227–234. doi: 10.1016/j.biologicals.2005.08.009. [DOI] [PubMed] [Google Scholar]
  • 14.Gu CJ, Zheng CY, Zhang Q, Shi LL, Li Y, Qu SF. An antiviral mechanism investigated with ribavirin as an RNA virus mutagen for foot-and-mouth disease virus. BMB Rep. 2006;39(1):9–15. doi: 10.5483/bmbrep.2006.39.1.009. [DOI] [PubMed] [Google Scholar]
  • 15.Habiela M, Seago J, Perez-Martin E, Waters R, Windsor M, Salguero FJ, Wood J, Charleston B, Juleff N. Laboratory animal models to study foot-and-mouth disease: a review with emphasis on natural and vaccine-induced immunity. J Gen Virol. 2014;95(Pt 11):2329. doi: 10.1099/vir.0.068270-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Harvie P, Omar RF, Dusserre N, Désormeaux A, Gourde P, Tremblay M, Beauchamp D, Bergeron MG. Antiviral efficacy and toxicity of ribavirin in murine acquired immunodeficiency syndrome model. J Acquir Immune DeficSyndr. 1996;12(5):451–461. doi: 10.1097/00042560-199608150-00003. [DOI] [PubMed] [Google Scholar]
  • 17.Herod MR, Adeyemi OO, Ward J, Bentley K, Harris M, Stonehouse NJ, Polyak SJ. The broad-spectrum antiviral drug arbidol inhibits foot-and-mouth disease virus genome replication. J Gen Virol. 2019;100(9):1293–1302. doi: 10.1099/jgv.0.001283. [DOI] [PubMed] [Google Scholar]
  • 18.Kim SM, Park JH, Lee KN, Kim SK, Ko YJ, Lee HS, Cho IS. Enhanced inhibition of foot-and-mouth disease virus by combinations of porcine interferon-α and antiviral agents. Antivir Res. 2012;96(2):213–220. doi: 10.1016/j.antiviral.2012.09.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Lefebvre DJ, Neyts J, De Clercq K. Development of a foot-and-mouth disease infection model in severe combined immunodeficient mice for the preliminary evaluation of antiviral drugs. Transbound Emerg Dis. 2010;57(6):430–433. doi: 10.1111/j.1865-1682.2010.01169.x. [DOI] [PubMed] [Google Scholar]
  • 20.Li ZH, Li CM, Ling P, Shen FH, Chen SH, Liu CC, Yu CK, Chen SH. Ribavirin reduces mortality in enterovirus 71–infected mice by decreasing viral replication. J Infect Dis. 2008;197(6):854–857. doi: 10.1086/527326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Lin P, Gao L, Huang Y, Chen Q, Shen H. An enterovirus 71 strain causes skeletal muscle damage in infected mice. Int J Clin Exp Pathol. 2015;8(4):3460. [PMC free article] [PubMed] [Google Scholar]
  • 22.Marcelin JR, Wilson JW, Razonable RR. Mayo clinic hematology/oncology and transplant infectious diseases services. Oral ribavirin therapy for respiratory syncytial virus infections in moderately to severely immunocompromised patients. Transpl Infect Dis. 2014;16(2):242–250. doi: 10.1111/tid.12194. [DOI] [PubMed] [Google Scholar]
  • 23.Molinari P, García-Nuñez S, Gravisaco MJ, Carrillo E, Berinstein A, Taboga O. Baculovirus treatment fully protects mice against a lethal challenge of FMDV. Antivir Res. 2010;87(2):276–279. doi: 10.1016/j.antiviral.2010.05.008. [DOI] [PubMed] [Google Scholar]
  • 24.Platt H. A study of the pathological changes produced in young mice by the virus of foot-and-mouth disease. J PatholBacteriol. 1956;72(1):299–312. doi: 10.1002/path.1700720135. [DOI] [PubMed] [Google Scholar]
  • 25.Rahman AU, Dhama K, Ali Q, Raza MA, Chaudhry U, Shabbir MZ. Foot and mouth disease in a wide range of wild hosts: a potential constraint in disease control efforts worldwide particularly in disease-endemic settings. Acta Trop. 2020 doi: 10.1016/j.actatropica.2020.105567. [DOI] [PubMed] [Google Scholar]
  • 26.Reed LJ, Muench H. A simple method of estimating fifty per cent endpoints. Am J Epidemiol. 1938;27(3):493–497. doi: 10.1093/oxfordjournals.aje.a118408. [DOI] [Google Scholar]
  • 27.Robinson L, Knight-Jones TJ, Charleston B, Rodriguez LL, Gay CG, Sumption KJ, Vosloo W. Global foot-and-mouth disease research update and gap analysis: 5–biotherapeutics and disinfectants. TransboundEmerg Dis. 2016;63:49–55. doi: 10.1111/tbed.12519. [DOI] [PubMed] [Google Scholar]
  • 28.Russmann S, Grattagliano I, Portincasa P, Palmieri VO, Palasciano G. Ribavirin-induced anemia: mechanisms, risk factors and related targets for future research. Curr Med chem. 2006;13(27):3351–3357. doi: 10.2174/092986706778773059. [DOI] [PubMed] [Google Scholar]
  • 29.Rweyemamu MM, Astudillo VM. Global perspective for foot and mouth disease control. Rev Off Int Epizoot. 2002;21(3):765–769. doi: 10.20506/RST.21.3.1378. [DOI] [PubMed] [Google Scholar]
  • 30.Salguero FJ, Sánchez-Martín MA, Díaz-San Segundo F, de Avila A, Sevilla N. Foot-and-mouth disease virus (FMDV) causes an acute disease that can be lethal for adult laboratory mice. Virology. 2005;332(1):384–396. doi: 10.1016/j.virol.2004.11.005. [DOI] [PubMed] [Google Scholar]
  • 31.Sanz-Ramos M, Díaz-San Segundo F, Escarmís C, Domingo E, Sevilla N. Hidden virulence determinants in a viral quasispecies in vivo. J Virol. 2008;82(21):10465–10476. doi: 10.1128/JVI.00825-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Sierra S, Dávila M, Lowenstein PR, Domingo E. Response of foot-and-mouth disease virus to increased mutagenesis: influence of viral load and fitness in loss of infectivity. J Virol. 2000;74(18):8316–8323. doi: 10.1111/tid.12194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Skinner HH. Propagation of strains of foot-and-mouth disease virus in unweaned white mice. Proc R Soc Med. 1951;1951:1041–1044. doi: 10.1177/003591575104401213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Skinner HH, Henderson WM, Brooksby JB. Use of unweaned white mice in foot-and-mouth disease research. Nature. 1952;169(4306):794–795. doi: 10.1038/169794a0. [DOI] [PubMed] [Google Scholar]
  • 35.Soumajit S, Selvan RP, Bhanuprakash V. In vitro antiviral efficacy of pleconaril and ribavirin on foot-and-mouth disease virus replication. Virusdisease. 2019;30(4):562–70. doi: 10.1007/s13337-019-00559-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Subak-Sharpe H. The effect of passage history, route of inoculation, virus strain and host strain on the susceptibility of adult mice to the virus of foot-and-mouth disease. Arch GesamteVirusforsch. 1961;11(3):373–399. doi: 10.1007/BF01249593. [DOI] [PubMed] [Google Scholar]
  • 37.Subak-Sharpe H, Pringle CR, Hollom SE. Factors influencing the dynamics of the multiplication of foot-and-mouth disease virus in adult mice. Archiv Arch ges Virusforsch. 1963;12(5):600–619. doi: 10.1007/BF01246383. [DOI] [PubMed] [Google Scholar]
  • 38.De la Torre JC, Alarcón B, Martinez-Salas E, Carrasco L, Domingo E. Ribavirin cures cells of a persistent infection with foot-and-mouth disease virus in vitro. J Virol. 1987;61(1):233–235. doi: 10.1128/JVI.61.1.233-235.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Weaver GV, Domenech J, Thiermann AR, Karesh WB. Foot and mouth disease: a look from the wild side. J Wildl Dis. 2013;49(4):759–785. doi: 10.7589/2012-11-276. [DOI] [PubMed] [Google Scholar]
  • 40.Zeng J, Wang H, Xie X, Li C, Zhou G, Yang D, Yu L. Ribavirin-resistant variants of foot-and-mouth disease virus: the effect of restricted quasispecies diversity on viral virulence. J Virol. 2014;88(8):4008–4020. doi: 10.1128/JVI.03594-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Zeng J, Wang H, Xie X, Yang D, Zhou G, Yu L. An increased replication fidelity mutant of foot-and-mouth disease virus retains fitness in vitro and virulence in vivo. Antivir Res. 2013;100(1):1–7. doi: 10.1016/j.antiviral.2013.07.008. [DOI] [PubMed] [Google Scholar]
  • 42.Zhang Z, Dong Z, Wei Q, Carr MJ, Li J, Ding S, Tong Y, Li D, Shi W. A neonatal murine model of coxsackievirus A6 infection for evaluation of antiviral and vaccine efficacy. J Virol. 2017 doi: 10.1128/JVI.02450-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Zhang G, Zhou F, Gu B, Ding C, Feng D, Xie F, Wang J, Zhang C, Cao Q, Deng Y, Hu W. In vitro and in vivo evaluation of ribavirin and pleconaril antiviral activity against enterovirus 71 infection. Arch Virol. 2012;157(4):669–679. doi: 10.1007/s00705-011-1222-6. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary file1 (MP4 4359 kb) (4.3MB, mp4)
Supplementary file2 (MP4 4711 kb) (4.6MB, mp4)
Supplementary file3 (MP4 4087 kb) (4MB, mp4)
Supplementary file4 (MP4 8271 kb) (8.1MB, mp4)
Supplementary file5 (MP4 3908 kb) (3.8MB, mp4)
Supplementary file6 (MP4 3414 kb) (3.3MB, mp4)
Supplementary file7 (MP4 4616 kb) (4.5MB, mp4)
Supplementary file8 (MP4 6577 kb) (6.4MB, mp4)
Supplementary file9 (MP4 4608 kb) (4.5MB, mp4)
Supplementary file10 (MP4 4649 kb) (4.5MB, mp4)

Articles from VirusDisease are provided here courtesy of Springer

RESOURCES