Efficacy of Interferon α-2b and Ribavirin against West Nile Virus In Vitro

To the Editor: West Nile virus (WNV) infected humans in the Western Hemisphere for the first time in the late summer of 1999. During 1999 and 2000, nine deaths occurred among 80 patients with meningitis or encephalitis in New York City; Westchester County, New York; New Jersey; and Connecticut (1–3). Effective antiviral agents are unknown for infections caused by WNV. Odelola (4) described 83% survival of WNV-infected mice and eradication of virus from brain when 1.5 mg. of ribavirin was administered by intraperitoneal injection after virus inoculation. Survival of controls was 25%. More recently, Jordan et al. have reported inhibition of WNV by a relatively high concentration of ribavirin (200 _M) given after infection of human oligodendroglial cells in vitro (5). Shahar et al. (6) reported protection of fetal mouse spinal cord tissues with mouse alpha and beta interferon before inoculation with WNV. We tested human recombinant interferon alpha-2b and ribavirin for activity against WNV in a primate cell system similar to that used to measure the effect of these agents on Bovine viral diarrhea virus, a cultivatable, closely related surrogate for Hepatitis C virus. 
 
Vero cells were cultured at 37o and 5% CO2 in a 96-well microtiter plate. Approximately 13,000 cells were seeded in each well 24 hours before specific concentrations of ribavirin or interferon alpha-2b were added. Approximately 2.5 X 103 PFU of WNV isolated from Culex pipiens (7) was added 1.5-2 hours after or before the addition of interferon alpha-2b or ribavirin to Vero cells. Forty-four hours after treatment, a colorimetric proliferation assay was used to measure viable cells in each treated well according to the protocol of Promega (Madison, WI). Cells exposed to specific concentrations of antiviral compounds, but without WNV, were used as negative controls. 
 
Interferon alpha-2b was protective and therapeutic. Interferon alpha-2b inhibited viral cytotoxicity at low dosage when applied before or after infection of cells with WNV. For example, viral protection was observed from 3,000 U/mL to 188 U/mL 2 hours before infection of cells with WNV. Interferon alpha-2b was also therapeutic when applied after cells were infected with WNV. Virus-induced cytotoxicity was inhibited by concentrations of ≥5.9 U/mL when added 1.5 hours after infection (Figure). The optical density 490 values in these tests were significantly different (p<0.05, using Tukey HSD multiple comparison test) when compared with the uninfected cells. 
 
 
 
Figure 
 
Effect of varying concentrations of interferon α-2b (FN) on West Nile virus-infected Vero cells. The vertical axis represents a colorimetric assay of cellular lactic dehydrogenase, which is directly proportional to cell viability and proliferation. ... 
 
 
 
Ribavirin was protective but not therapeutic in vitro. Cells were protected at dosages of 400 and 500 _M but not at dosages of ≤300 _M of ribavirin applied 2 hours before infection of cells with WNV. A cytotoxic effect of ribavirin occurred at concentrations of 600-1,000 _M. 
 
In humans, daily doses of 3 million units of interferon result in serum levels of 10-20 U/mL, well above that required for in vitro efficacy (8). In contrast, oral ribavirin doses of 2,400 mg daily yield a steady-state serum concentration of 3-4 _g/mL after several days, approximately 12-40-fold less than the in vitro inhibitory concentrations of 200-500 _M (50-125 _g/mL) noted by Jordan et al. (5) and in this study. Intravenous administration of 4 g daily, as used in the treatment of Lassa fever, would be required to reach a potentially effective serum concentration (9,10). However, intracellular accumulation and phosphorylation of ribavirin may account for its therapeutic effect in mice (4). 
 
We conclude that interferon alpha-2b possesses greater therapeutic activity in vitro than ribavirin, with a potentially greater therapeutic ratio in humans. Whether combination therapy, as employed against hepatitis C, may be optimal requires further study.

On admission, the patient's fever was still high (40°C), there was severe hypoxemia with bilateral diffuse pulmonary infiltrates, a tachyarrhythmia with auricular fibrillation and gallop, and conjunctival injection. Laboratory results indicated mild renal insufficiency (urea 12.5 mmol/L; creatininemia 180 µmol/ L), hepatic cytolysis (serum glutamicoxaloacetic transaminase 236 and serum glutamic-pyruvic transaminase 72), a moderate thrombocytopenia (platelet counts 86 000/mm 3 ), an inflammatory syndrome (C-reactive protein 272 mg/L), and a capillary leak syndrome (hematocrit 49%; albuminemia 20 g/L). On the night after admission, an aggravation of the cardiac function with myocarditis developed; it responded quickly to symptomatic treatment. The patient's condition improved steadily on the following days with a reduction of the pulmonary manifestations, and he was discharged on April 2.
Blood samples obtained on March 21 and March 29 were tested for the presence of antibodies to hantaviruses (Puumala, Hantaan, Sin Nombre, [SNV] Seoul, and Laguna Negra) by immunoglobulin (Ig) M-capture and IgG enzyme-linked immunosorbent assay. IgM antibodies were detected for all these antigens on the first sample, but there was no increase on the second sample. A substantial increase in IgG titer for SNV and Laguna Negra antigens was observed from the first to the second sample, but not for the other antigens. The virus could not be detected either by reverse transcription-polymerase chain reaction or by inoculation into cell culture (three passages). Since the identification in 1993 of SNV as the cause of HPS (1), numerous cases of this disease have been confirmed in various regions of North and South America. The first HPS cases associated with Andes virus in Argentina (2) were observed in 1995. Since then, more than 500 HPS cases have been reported in six countries of South America (Argentina, Bolivia, Brazil, Chile, Paraguay, and Uruguay), with mortality rates ranging from 30% to 70%.
Hantaviruses are rodent-borne, and each is associated with a specific rodent. Sigmodontine rodents are the vectors of hantaviruses associated with HPS. Infections are most frequently transmitted by inhalation of virus-contaminated aerosols of rodent excreta, but human-tohuman transmission has also been described (3).
The patient described here was probably infected in Chile and more likely in the Puerto Monte area, where HPS cases were reported in 2001. Unfortunately, virus could not be detected because the first blood sample was obtained 2 weeks after onset of fever.  (6) reported protection of fetal mouse spinal cord tissues with mouse alpha and beta interferon before inoculation with WNV. We tested human recombinant interferon alpha-2b and ribavirin for activity against WNV in a primate cell system similar to that used to measure the effect of these agents on Bovine viral diarrhea virus, a cultivatable, closely related surrogate for Hepatitis C virus.Vero cells were cultured at 37 o and 5% CO 2 in a 96-well microtiter plate. Approximately 13,000 cells were seeded in each well 24 hours before specific concentrations of ribavirin or interferon alpha-2b were added. Approximately 2.5 X 10 3 PFU of WNV isolated from Culex pipiens (7) was added 1.5-2 hours after or before the addition of interferon alpha-2b or ribavirin to Vero cells. Forty-four hours after treatment, a colorimetric proliferation assay was used to measure viable cells in each treated well according to the protocol of Promega (Madison, WI). Cells exposed to specific concentrations of antiviral compounds, but without WNV, were used as negative controls.

Efficacy of Interferon
Interferon alpha-2b was protective and therapeutic. Interferon alpha-2b inhibited viral cytotoxicity at low dosage when applied before or after infection of cells with WNV. For example, viral protection was observed from 3,000 U/mL to 188 U/mL 2 hours before infection of cells with WNV. Interferon alpha-2b was also therapeutic when applied after cells were infected with WNV. Virusinduced cytotoxicity was inhibited by concentrations of >5.9 U/mL when added 1.5 hours after infection (Figure). The optical density 490 values in these tests were significantly different (p<0.05, using Tukey HSD multiple LETTERS comparison test) when compared with the uninfected cells. Ribavirin was protective but not therapeutic in vitro. Cells were protected at dosages of 400 and 500 µM but not at dosages of <300 µM of ribavirin applied 2 hours before infection of cells with WNV. A cytotoxic effect of ribavirin occurred at concentrations of 600-1,000 µM.
In humans, daily doses of 3 million units of interferon result in serum levels of 10-20 U/mL, well above that required for in vitro efficacy (8). In contrast, oral ribavirin doses of 2,400 mg daily yield a steady-state serum concentration of 3-4 µg/mL after several days, approximately 12-40-fold less than the in vitro inhibitory concentrations of 200-500 µM (50-125 µg/mL) noted by Jordan et al. (5) and in this study. Intravenous administration of 4 g daily, as used in the treatment of Lassa fever, would be required to reach a potentially effective serum concentration (9,10). However, intracellular accumulation and phosphorylation of ribavirin may account for its therapeutic effect in mice (4).
We conclude that interferon alpha-2b possesses greater therapeutic activity in vitro than ribavirin, with a potentially greater therapeutic ratio in humans. Whether combination therapy, as employed against hepatitis C, may be optimal requires further study.