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Am. J. Trop. Med. Hyg., 75(5), 2006, pp. 1011-1016
Copyright © 2006 by The American Society of Tropical Medicine and Hygiene

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IN VITRO AND IN VIVO STUDIES OF RIBAVIRIN ACTION ON BRAZILIAN ORTHOBUNYAVIRUS

MÁRCIA C. LIVONESI*, RICARDO L. MORO DE SOUSA, SORAYA J. BADRA, AND LUIZ T.M. FIGUEIREDO
Center for Research in Virology, School of Medicine of Ribeirão Preto, University of São Paulo USP, Ribeirão Preto, SP, Brazil


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oropouche, Caraparu, Guama, Guaroa, and Tacaiuma are viruses (genus Orthobunyavirus) that cause human febrile illnesses and encephalitis. The goal of this study was to evaluate the antiviral action of ribavirin on these orthobunyaviruses to achieve a therapeutical agent to treat the diseases caused by these viruses. In vitro results showed that ribavirin (50 µg/mL) had antiviral activity only on the Tacaiuma virus. Addition of guanosine in the culture reversed the antiviral effect of ribavirin on Tacaiuma virus, suggesting that ribavirin inhibited this virus by reducing the intra-cellular guanosine pool. Moreover, ribavirin was not an effective drug in vivo because it was unable to inhibit the death of the mice or virus replication in the brain. The results suggest that ribavirin has no antiviral activity on the Oropouche, Caraparu, Guama, Guaroa, or Tacaiuma viruses; consequently, ribavirin would not be a good therapeutical agent to treat these arboviruses.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Oropouche (OROV), Caraparu (CARV), Guama (GMAV), Guaroa (GROV), and Tacaiuma (TCMV) viruses belong to distinct antigenic serogroups of the genus Orthobu-nyavirus in the Bunyaviridae family. These viruses are enveloped with trisegmented single-stranded RNA genome of negative or ambisense polarity, replicate in the cytoplasm, and bud into the Golgi apparatus or upon the plasma membrane.1,2

OROV (Simbu group) is transmitted mainly by the biting midge (Culicoides paraensis) and has been associated with dengue-like acute febrile illness. OROV fever has emerged over the past 40 years as a serious public health problem in tropical and subtropical areas of Central and South America, having caused at least 30 reported outbreaks involving more than half a million people.3 Clinical features of OROV fever include abrupt onset of fever, chills, severe headache, dizziness, myalgia, arthralgia, nausea, and vomiting. Occasionally, neurologic involvement has been reported. All ages and both sexes appear to be equally susceptible to infection.4 Similarly to OROV, CARV (C group), GMAV (Guama group), GROV (California encephalitis and Bunyamwera group), and TCMV (Anopheles A group) have also been associated with febrile illness in humans. They are transmitted by mosquitoes and cause disease mainly in residents of the Amazon region of Brazil.5–8 Due to the high attack rates of OROV epidemics and to the debilitating nature and duration of symptoms of the clinical syndromes caused by this and other Brazilian orthobunyaviruses, antiviral therapy, if available, would become a very helpful intervention.

Ribavirin (RBV), the nucleoside analogue 1-ß-D-ribo-furanosyl-1,2,4-triazole-3-carboxamide, exhibits antiviral activity against a variety of RNA viruses in cell culture,9,10 including viruses from the Paramyxoviridae,11,12 Flaviviri-dae,12–14 Picornaviridae,15 Orthomyxoviridae,16 Arenaviri-dae,17,18 and Bunyaviridae19,20 families. In humans, RBV is used clinically to treat infections by hepatitis C virus (in combination with interferon-{alpha}),21,22 respiratory syncytial virus,23 and Lassa fever virus.24 RBV is phosphorylated by cellular enzymes and has been proposed to exert antiviral effects through several mechanisms25: (A) reduction in cellular gua-nosine triphosphate (GTP) pools via inhibition of inosine monophosphate dehydrogenase (IMPDH),26 (B) inhibition of the viral RNA guanylyltransferase and, consequently, reduction of the capping of viral mRNA,27 (C) inhibition of the viral RNA polymerase,28 (D) incorporation of the compound either as a GTP or an ATP analogue, causing lethal mutagen-esis of the viral RNA genomes,15 and (E) enhancement of the Th1 antiviral immune response.29 RBV monophosphate is the derivative responsible for the first mechanism, and RBV triphosphate is linked to the second, third, and fourth of these mechanisms.30

In an effort to characterize antiviral agents that could attenuate infection caused by OROV, CARV, GMAV, GROV, and TCMV, we tested the action of RBV, a broad-spectrum antiviral drug, on these viruses both in vitro and in vivo.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Viruses. ORO (BeAn19991), GUA (BeAn277), GRO (BeH22063), and TCM (BeAn73) viruses were kindly supplied by Dr. Pedro Vasconcelos and Dr. Amélia Travassos da Rosa, (Evandro Chagas Institute, Brazilian Ministry of Health, Belém, Brazil, and University of Texas Medical Branch, Galveston, Texas). CARV (SPAn2049) was kindly supplied by Dr. Terezinha Lisieux Coimbra (Adolpho Lutz Institute, São Paulo, Brazil). Viral stocks were obtained from the brains of intracerebrally infected suckling mice. Brains were mixed with PBS (dilution 1:10 wt/vol), macerated, and centrifuged at 2000 x g for 10 minutes at 4°C. The supernatants were harvested and stored at -70°C until use.

Cell culture. African green monkey kidney (Vero E6) cells (ATCC-CCL81) were grown in minimum essential medium (MEM, Cultilab, Brazil) supplemented with 10% inactivated, Mycoplasma-free, fetal bovine serum (FBS, Cultilab), 1% L-glutamine, and 0.3% sodium bicarbonate.

Compounds. RBV and guanosine were purchased commercially (Sigma Chemical Co., St. Louis, MO). Ribavirin was diluted in 0.85% NaCl solution, and guanosine (G) was diluted in 30% ethanol, and they were stored at 4°C until use.

Animals. Swiss newborn mice were obtained from the laboratory animal facility of the University of São Paulo (Ribeirão Preto, Brazil). The mice were maintained in microisolator cages in the animal housing facility of the Center for Research in Virology (University of São Paulo, Ribeirão Preto, Brazil). The experiments were approved by the ethical committee on vertebrate animal experiments of the University of São Paulo (no. 006/2004).

In vitro antiviral evaluation. In vitro antiviral evaluation was done with a plaque assay. Vero E6 cells were seeded in 24-well plates in MEM with 10% FBS, for 24 hours at 37°C and 5% CO2. Medium was removed, serial 10-fold dilutions of viral stocks diluted in MEM with 5% FBS were added (0.2 mL/well) in quadruplicate, and the cells were incubated for 2 hours at 37°C. Subsequently, the viral inoculum was removed, and 1.0 mL of a combination (vol/vol) of 1% low-melting-point agarose plus 2x MEM (10% FBS) was added to each well; the plates were incubated at 37°C for 3 days for OROV and GMAV, 5 days for CARV and GROV, and 9 days for TCMV. The plaques were visualized by staining with a naph-thol blue black solution (15 minutes) after removal of the agarose plug.31 The plaques were counted under an inverted microscope, and the virus titer was determined as Log10 PFU per milliliter.

Ribavirin and guanosine were diluted in the medium and added to cells on the day before, or 2 hours after, viral infection. Virus titers obtained in the presence and absence of RBV were compared, and the results were plotted as percentage of inhibition on plaque formation (Table 1Go).


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TABLE 1
Effect of RBV on Brazilian Orthobunyavirus replication in Vero E6 cells
 
The concentration of RBV added to the cell cultures was ≤50 µg/mL because this concentration had a mild cytostatic effect, inhibiting Vero E6 cell growth by 50%.32 Moreover, clinically therapeutic levels of RBV are 25 µg/mL (or 100 µM).33 Exogenous guanosine was added to the cell cultures at a concentration of 30 µg/mL.

Determination in vivo of RBV toxicity. Ribavirin toxicity was evaluated by significant weight loss, which is a sign of RBV-induced anemia. RBV doses used were 45 and 35 mg/ kg/day. The mice were treated intraperitoneally (IP) daily for 10 days. The animal weights were determined prior to the first treatment and daily for 10 days.

Intraperitoneal challenge with viruses and administration of RBV. Three-day-old Swiss mice were infected IP with OROV (10 LD50), CARV (1,000 LD50), GMAV (100 LD50), GROV (100 LD50), or TCMV (1,000 LD50) in a volume of 40 µL per mouse. The mice were treated intraperitoneally with RBV or placebo in a volume of 30 µL per mouse. The treatment was initiated 24 hours before infection and maintained every day. The animals were monitored daily for mortality.

Determination of brain virus titers. The brains of mice (two mice per group) were taken aseptically on days 1, 2, 3, 5, 7, and 9 after infection. Brains were mixed with PBS (dilution 1:10 wt/vol), macerated, and centrifuged at 2000 x g for 10 minutes at 4°C. Supernatants were harvested and stored at –70°C before plaque assay on Vero E6 cells. Virus titers in the brain were expressed as Log10 PFU/mL.

Statistical analysis. Analysis of variance, followed by the parametric Tukey-Kramer test was used in the in vitro experiments. Student’s t-test was used to determine if there was a significant difference in the body weight of the mouse treated with different doses of RBV or placebo and virus titers between the treated and the placebo groups. A P value < 0.05 was considered to indicate statistical significance.


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In vitro antiviral effect of RBV. RBV (50 µg/mL) presented a significant inhibitory effect on TCMV replication, either 24 hours before (inhibition 85%; P < 0.005) or 2 hours after (inhibition 62%; P < 0.01) infection in Vero E6 cells. Moreover, RBV showed a weak inhibitory effect on GUAV replication when treatment was initiated 24 hours prior to infection. On the other hand, RBV was unable to inhibit replication of other viruses tested (Table 1Go).

To determine whether doses lower than 50 µg/mL could inhibit TCMV replication, cells were treated 1 day before (Figure 1AGo) or 2 hours after (Figure 1BGo) infection with doses ≤ 50 µg/mL. Figure 1Go shows that only the concentration of 50 µg/mL is able to significantly inhibit TCMV replication. Moreover, the concentration of 50 µg/mL has an antiviral effect only when treatment is initiated early; treatment 24 and 48 hours after infection does not have an inhibitory effect on TCMV replication (Figure 1CGo).


Figure 1
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  		    FIGURE 1. RBV has an antiviral effect on TCMV only at a dose of 50 µg/mL, and this is abolished by addition of guanosine. (A, B) Vero E6 cells cultured in 24-well plates and treated only with medium (M) or with different concentrations of RBV (≤ 50 µg/mL) 24 hr before (A) or 2 hr after (B) infection by TCMV. (C) Vero E6 cells treated only with medium (M) or with medium added to RBV (50 µg/mL) 24 or 48 hours after infection by TCMV. (D) Vero E6 cells cultured in 24-well plates and treated only with medium (M), M added to guanosine (G), or M added to RBV, or M plus RBV and G 24 hr before infection by TCMV. (A–D) The overlay was removed 9 days after infection, cells were stained with naphthol blue black, and plaque-forming units (PFU) were counted. Scale bars represent the mean ± SD of Log10 PFU/mL obtained from quadruplicate cultures. Similar results were obtained in a second experiment. *P < 0.05 compared with medium-treated infected cells.

 
To investigate whether the inhibition of TCMV replication by RBV was caused by depletion of the GTP pool, exogenous guanosine was added to the culture medium at concentration of 30 µg/mL. Figure 1DGo shows that addition of guanosine efficiently reversed the inhibitory effect of RBV on the TCMV replication in Vero E6 cells, suggesting that the antiviral activity of RBV on TCMV is indeed related to reduction in intracellular GTP levels.

In vivo antiviral effect of RBV. To determine the maximum nontoxic dose, suckling mice were treated with placebo or with 35 or 45 mg/kg, of a single daily dose of RBV for 10 days. Figure 2Go shows that the dose of 35 mg/kg/day was well tolerated by mice because they presented an increase of weight similar to the placebo-treated mice. On the other hand, mice treated with 45 mg/kg/day had significant weight loss beginning on day 7 of treatment (P < 0.05) when compared with placebo-treated mice, demonstrating that this dose is toxic to suckling mice. Thus, RBV at 35 mg/kg/day was the dose chosen to treat the mice infected by viruses ORO, CAR, GMA, GRO, and TCM.


Figure 2
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  		    FIGURE 2. RBV at 35 mg/kg/day is not toxic in suckling mice. Groups of Swiss mice were treated daily with placebo or 45 or 35 mg/kg of RBV, for 10 consecutive days. Treatment was initiated on 2-day-old mice (black arrow) and was maintained for 10 days. The animals were weighed before treatment and daily for 10 days. Values represent the mean of mouse weights of each group. *P < 0.05 compared with placebo-treated mice. Similar results were obtained in a second experiment.

 
RBV treatment consisting of a single daily dose was initiated 1 day before the viral infection until the death of placebo-treated mice. Intraperitoneal administration of RBV did not prevent the death of mice infected by studied orthobun-yaviruses (Table 2Go), although RBV-treated mice infected by OROV or CARV presented survival rates of 6% and 12%, respectively, which was not significant (Table 2Go). Likewise, RBV treatment did not result in a significant increase in the mean time to death of infected mice (Table 2Go), demonstrating that RBV had no in vivo antiviral action on OROV, CARV, GMAV, GROV, or TCMV.


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TABLE 2
Effect of RBV on mice infected i.p. with Brazilian Orthobunyavirus
 
Next, we studied the effect of RBV treatment on virus migration and replication in brain tissue. Figure 3Go shows that RBV treatment prevented neither migration nor replication of the studied orthobunyaviruses; the viruses appeared in the brains of the two groups in the same period, and the virus titer in the brain was equivalent between both RBV-treated and placebo-treated groups. These data explain the ineffectiveness of RBV in preventing the death of virus-infected mice and confirm that RBV does not have antiviral activity in vivo on the studied viruses.


Figure 3
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  		    FIGURE 3. RBV-treated and placebo-treated mice present similar virus titers in the brain. Groups of 16 three-day-old Swiss mice were infected intraperitoneally with OROV, CARV, GMAV, GROV, or TCMV, and they were treated intraperitoneally with RBV (35 mg/kg/day) or placebo. Treatment was initiated 24 hours before infection and maintained each every day. Mouse brains (two mice per group) were taken on days 1, 2, 3, 5, 7, and 9 after infection. Virus titer in the brain was measured by plaque assay. Scale bars represent the mean ± SD of PFU/mL. Similar results were obtained in a second experiment.

 

DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, RBV was evaluated for its antiviral action on OROV, CARV, GMAV, GROV, and TCMV in vitro and in vivo. RBV had a significant antiviral effect in vitro, only on TCMV, both before and after viral infection (Table 1Go). However, this antiviral effect was abolished when the treatment started 24 hours after infection (Figure 1CGo) or when we used concentrations < 50 µg/mL (Figure 1. A and BGo), suggesting that use of the therapeutic level of RBV, which is 25 µg/mL, could be ineffective to treat illness caused by TCMV. This supposition is confirmed by experiments in vivo, where the RBV did not show any antiviral effect on OROV, CARV, GMAV, GROV, or TCMV. Thus, RBV was unable to prevent death of infected mice, it did not prolong the mean time to death (Table 2Go), and it did not prevent virus migration and replication in brain tissue (Figure 3Go).

The inhibitory effect of RBV on TCMV in vitro was reversed by addition of guanosine (Figure 1DGo), suggesting that the mechanisms of these antiviral actions, in this model, were mediated through depletion of intracellular GTP, which could hypothetically reduce the efficiency of the viral polymerase activity. However, RBV had no inhibitory effect on OROV, CARV, GMAV, or GROV replication (Table 1Go), suggesting that the replicative cycle of these viruses is less dependent on intracellular GTP levels. Schiedel and others30 produced RBV-resistant Sindbis virus (family Togaviridae, genus Al-phavirus) by performing serial passages of the virus in A. albopictus cells in the presence of MPA (mycophenolic acid). The authors suggested that Sindbis virus mutants were able to replicate in the presence of RBV and MPA because they generated a guanylyltransferase and/or RNA polymerase with an increased GTP affinity. Subsequently, Schiedel and others34 showed that the MPA-resistant Sindbis virus had an alteration on the RNA guanylyltransferase. Guanylyltrans-ferase is an important protein for the 5'-cap structure formation of cellular and viral mRNAs. However, viruses of the Bunyaviridae family do not encode enzymes for making 5'-cap of mRNAs because they have a mechanism known as cap-snatching where they use host-derived primers to initiate the mRNA transcription process.35 Thus, resistance to RBV observed in vitro by OROV, CARV, GMAV, GROV, and TCMV (when doses < 50 µg/mL were used, and treatment starting 24 hours post-infection) is not associated with guany-lyltransferase protein, but it could be related to some particularity of the RNA polymerase of these viruses. A similar in vitro result was reported on absence of action of RBV on SARS-CoV (severe acute respiratory syndrome-coronavirus). However, the RBV resistance mechanism has not been explained by the authors.36 Moreover, RBV-resistant variants of HCV and poliovirus have been isolated from patients treated with RBV monotherapy.37–39 In these studies, resistance to RBV was associated with amino acid substitution in the RdRp (RNA-dependent RNA polymerase) of these viruses.37–39 These data corroborate the assumption that the resistance of OROV, CARV, GMAV, GROV, and TCMV to RBV antiviral action could be related to some characteristic of the RNA polymerase of these viruses. However, this has not been evaluated in the present study.

Intraperitoneal inoculation of virus produces first a systemic disease; later, the viruses migrate to the brain, where they replicate and cause the death of suckling mice (data not showed). Thus, we can propose that the RBV resistance in vivo by the OROV, CARV, GMAV, GROV, and TCMV can be associated with two factors: first, because of some peculiarity of the RNA polymerase of these viruses, which was not inhibited by RBV action and permitted virus replication with consequent increase viral burden, the virus consequently reached the brains of the mice, causing illness and death; second, RBV was not seen to be effective against intracere-bral virus infections of mice unless it was administered directly into the brain,40 suggesting that RBV or its active me-tabolites do not reach the brain in adequate concentrations, probably because the RBV does not cross the blood-brain barrier.41 When the viruses reach the brain, however, they can replicate in an environment free of the antiviral action of RBV, causing disease and death in the infected animals. This second factor was described in mice infected with dengue virus, treatment of which with RBV was ineffective but treatment with a lipophilic analogue of RBV was more effective because it crossed the blood-brain barrier.41 A previous in vivo study showed that the CARV was susceptible to antiviral action of RBV, and this is at variance with our results.5 A possible explanation is based on the experimental model used. In that study, 4- to 6-week-old B6C3F1 mice were inoculated intraperitoneally with CARV and died after they had developed coagulative liver necrosis. However, when B6C3F1 mice were CARV-infected and treated with RBV, the liver necrosis was not observed and consequently, the mice were protected from death, possibly because RBV would inhibit the viral replication in the hepatic tissue. However, in our experimental model, suckling mice died of encephalitis (Figure 3Go), possibly because RBV did not cross the blood-brain barrier and consequently could not inhibit viral replication in the cerebral tissue, suggesting that the RBV would not present antiviral action on CARV during episodes of encephalitis.

In conclusion, our results indicate that RBV does not present antiviral action on OROV, CARV, GMAV, GROV, or TCMV; consequently, RBV will not be a good therapeutical agent to treat these arboviruses. Future studies will be necessary to characterize other antiviral agents to attenuate the infection caused by these viruses.

Received April 19, 2006. Accepted for publication July 21, 2006.

Acknowledgments: We thank Dr. Pedro Vasconcelos, Dr. Amélia Travassos da Rosa, and Dr. Terezinha Lisieux Coimbra for kindly supplying the viruses used in this study and Dr. Eurico de Arruda Neto for his critical review of the manuscript.

Financial Support: This work was supported by an FAPESP grant to L.T.M. Figueiredo (03/03682-3) and by a fellowship from CAPES to M.C. Livonesi.

* Address correspondence to Márcia C. Livonesi, Center for Research in Virology, School of Medicine of Ribeirão Preto, University of São Paulo USP, Ribeirão Preto, SP, Brazil. E-mail: pink{at}rpm.fmrp.usp.br Back

Authors’ addresses: Márcia C. Livonesi, Ricardo L.M. de Sousa, Sor-aya J. Badra, and Luiz T.M. Figueiredo, Centro de Pesquisa em Vi-rologia, Universidade de São Paulo (USP), Av. Bandeirantes, 3900, 14049-900, Ribeirão Preto, SP, Brasil, Telephone/Fax: +55-16-602-3376.

Reprint requests: Luiz Tadeu Moraes Figueiredo, Centro de Pesquisa em Virologia, Faculdade de Medicina de Ribeirão Preto, Univer-sidade de São Paulo (USP), Av. Bandeirantes, 3900, CEP 14049-900, Ribeirão Preto, SP, Brasil. E-mail: ltmfigue{at}fmrp.usp.br.


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