In vitro and in vivo activity of ribavirin against Andes virus infection

Abstract

Pathogenic hantaviruses are a closely related group of rodent-borne viruses which are responsible for two distinct diseases in humans, hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome (HPS, otherwise known as hantavirus cardiopulmonary syndrome, HCPS). The antiviral effect of ribavirin against Old World hantaviruses, most notably Hantaan virus, is well documented; however, only a few studies have addressed its inhibitory effect on New World hantaviruses. In the present study, we demonstrate that ribavirin is highly active against Andes virus (ANDV), an important etiological agent of HPS, both in vitro and in vivo using a lethal hamster model of HPS. Treatment of ANDV infected Vero E6 cells with ribavirin resulted in dose-dependent reductions in viral RNA and protein as well as virus yields with a half maximal inhibitory concentration between 5 and 12.5 µg ml(-1). In hamsters, treatment with as little as 5 mg kg(-1) day(-1) was 100% effective at preventing lethal HPS disease when therapy was administered by intraperitoneal injection from day 1 through day 10 post-infection. Significant reductions were observed in ANDV RNA and antigen positive cells in lung and liver tissues. Ribavirin remained completely protective when administered by intraperitoneal injections up to three days post-infection. In addition, we show that daily oral ribavirin therapy initiated 1 day post-infection and continuing for ten days is also protective against lethal ANDV disease, even at doses of 5 mg kg(-1) day(-1). Our results suggest ribavirin treatment is beneficial for postexposure prophylaxis against HPS-causing hantaviruses and should be considered in scenarios where exposure to the virus is probable. The similarities between the results obtained in this study and those from previous clinical evaluations of ribavirin against HPS, further validate the hamster model of lethal HPS and demonstrate its usefulness in screening antiviral agents against this disease.

Figure 1. In vitro effect of ribavirin against Andes virus.

Vero E6 cells were infected with Andes virus (m.o.i. = 0.01) and cultured in the presence of varying concentrations of ribavirin. (A) Supernatants collected at 3, 5 and 7 days post-infection were titered using a focus forming unit (FFU) assay. Day 1 supernatants were also collected, though titers were below the limit of detection of the focus assay. Data represent the average of three measurements. Error bars represent the standard error of the mean. (B–C) Andes virus RNA concentrations in supernatant (B) and Vero E6 cells (C) were determined using quantitative real-time RT-PCR targeting the nucleoprotein coding region. Error bars represent the standard error of the mean. (D) Andes virus nucleoprotein expression levels in infected Vero E6 cells cultured in the presence of ribavirin for 7 days post-infection. * p<0.05, ** p<0.01,*** p<0.0001.

Figure 2. Effect of daily ribavirin therapy on lethal Andes virus infection in hamsters.

Groups of 12 hamsters were infected with a lethal dose of Andes virus and treated by daily intraperitoneal injections of 0, 5, 25, 50 or 100 mg ribavirin kg⁻¹ day⁻¹ from day 1 through day 10 post-infection. For each group, 6 hamsters were monitored for survival (A), while 3 hamsters per group were euthanized on days 6 and 8 post-infection and samples collected for virological analysis (B–C). Andes virus RNA profiles were determined in lung and liver (B) and blood (C) samples collected from hamsters as noted above. The data represents the average values obtained from triplicate analysis of tissue samples collected from three hamsters per treatment group, per time point. Error bars represent the standard error of the mean.

Figure 3. Histological analysis of lungs from Andes virus infected ribavirin treated and control hamsters.

Hamsters were infected with a lethal dose of Andes virus and treated daily with ribavirin at the indicated concentrations from day 1 through day 10 post-infection. Shown are hematoxylin and eosin (H&E) and immunohistochemistry (IHC) stained sections of lungs collected at day 8 post-infection from treated and control hamsters. Histological abnormalities were only noted in control hamsters which demonstrated perivascular edema (see *). IHC with a monoclonal antibody (shown) and polyclonal sera (not shown) revealed drastic reductions in the detection of Andes virus nucleoprotein in all ribavirin treated hamsters compared with the diffuse staining noted in the lung endothelium of control (PBS) treated animals.

Figure 4. Analysis of host responses in ribavirin treated and control animals.

Hamsters were infected with a lethal dose of Andes virus and treated daily with ribavirin at the indicated concentrations administered by intraperitoneal injections. Host responses to infection were monitored in lung samples collected at 8 days post-infection using recently developed, hamster specific, real-time RT-PCR assays. Shown are the average fold changes of triplicate analysis of samples collected from 3 hamsters per treatment group. Data is normalized to uninfected animals and error bars represent the standard error of the mean.

Figure 5. Effect of abbreviated or delayed ribavirin therapy on lethal Andes virus infection in hamsters.

Hamsters were infected with a lethal dose of Andes virus as described in the materials and methods section and treated with ribavirin at 50 mg kg⁻¹ day⁻¹ as outlined below. (A) Effect of abbreviated ribavirin therapy. Groups of 6 (treated) or 3 (control) infected hamsters were treated with ribavirin or PBS beginning on day 1 post-infection. On days 3, 5 and 7 post-infection therapy was terminated for one treatment and control group. (B) Effect of delayed ribavirin therapy. On days 3, 5 and 7 post-infection, a ten day therapeutic schedule consisting of daily ribavirin or PBS injections was initiated for one treatment or control group (6 and 3 infected hamsters, respectively).

Figure 6. Protective efficacy of oral ribavirin therapy. (A) Effect of oral ribavirin therapy.

Hamsters were infected with Andes virus as outlined in the material and methods section and treated with oral ribavirin at concentrations of 0, 5 or 50 mg kg⁻¹ day⁻¹ from day 1 through day 10 post-infection. (B) Chest radiographs of ribavirin treated and untreated hamsters. Extensive fulminant, bilateral lung infiltrates were observed at day 8 post-infection in control hamsters, whereas ribavirin treated hamsters showed little to no infiltrations. Shown are representative chest x-rays (ventral dorsal) from hamsters treated orally with 0, 5 or 50 mg ribavirin kg⁻¹ day⁻¹.