Treatment with interferon-α2b and ribavirin improves outcome in MERS-CoV–infected rhesus macaques

Journal name:
Nature Medicine
Volume:
19,
Pages:
1313–1317
Year published:
DOI:
doi:10.1038/nm.3362
Received
Accepted
Published online

Abstract

The emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) is of global concern: the virus has caused severe respiratory illness, with 111 confirmed cases and 52 deaths1 at the time of this article's publication. Therapeutic interventions have not been evaluated in vivo; thus, patient management relies exclusively on supportive care, which, given the high case-fatality rate, is not highly effective. The rhesus macaque is the only known model organism for MERS-CoV infection, developing an acute localized to widespread pneumonia with transient clinical disease2, 3 that recapitulates mild to moderate human MERS-CoV cases4, 5. The combination of interferon-α2b and ribavirin was effective in reducing MERS-CoV replication in vitro6; therefore, we initiated this treatment 8 h after inoculation of rhesus macaques. In contrast to untreated, infected macaques, treated animals did not develop breathing abnormalities and showed no or very mild radiographic evidence of pneumonia. Moreover, treated animals showed lower levels of systemic (serum) and local (lung) proinflammatory markers, in addition to fewer viral genome copies, distinct gene expression and less severe histopathological changes in the lungs. Taken together, these data suggest that treatment of MERS-CoV infected rhesus macaques with IFN-α2b and ribavirin reduces virus replication, moderates the host response and improves clinical outcome. As these two drugs are already used in combination in the clinic for other infections, IFN-α2b and ribavirin should be considered for the management of MERS-CoV cases.

At a glance

Figures

  1. Schedule of treatment and selected clinical laboratory parameters.
    Figure 1: Schedule of treatment and selected clinical laboratory parameters.

    (a) Schematic showing the experiment schedule. IFN-α2b was delivered subcutaneously at 5 MIU/kg. Ribavirin was delivered as an initial loading dose intravenously at 30 mg/kg (full circle) with subsequent doses delivered intramuscularly at 10 mg/kg (half-full circle). Rhesus macaques were given either IFN-α2b and ribavirin (treated, RM1–RM3, n = 3) or sham treatment (untreated, RM4–RM6, n = 3) on the same schedule. (b) Clinical score (see also Supplementary Table 1) determined from an established scoring sheet34. (c) Changes in oxygen saturation from preinoculation values (% δSPO2). (d,e) The total number of white blood cells (WBCs) (d) and neutrophils (e) in blood samples obtained from animals 0, 24, 48 and 72 h after infection. All values are mean ± s.d. (two-way analysis of variance (ANOVA), Bonferroni's post hoc test, **P < 0.01). A single sample per time point per animal was used for the analysis. K, cell count × 103.

  2. Radiographic alterations.
    Figure 2: Radiographic alterations.

    Ventrodorsal thoracic X-rays from IFN-α2b– and ribavirin–treated (RM1–RM3) and untreated (RM4–RM6) rhesus macaques imaged before MERS-CoV infection (day 0) and on day 3 after infection. Areas of interstitial infiltration, indicative of pneumonia, are circled. R indicates the right side of the animal.

  3. Pathology and viral loads in selected tissue samples.
    Figure 3: Pathology and viral loads in selected tissue samples.

    (a) Gross pathology of lungs from IFN-α2b– and ribavirin–treated (left) and untreated (middle) MERS-CoV–infected rhesus macaques 72 h after inoculation. The percentage area showing gross pathology is indicated on the right. Six lung lobes per animal (dorsal and ventral) from three animals per group were scored. (b) The mean viral load determined by qRT-PCR from individual tissues collected at necropsy. The mean viral load of all lung lobes combined is indicated in the inset. Log TCID50 eq/g, log TCID50 equivalents per gram tissue; one sample per tissue per animal from three animals per group were analyzed. (c,d) Lung tissue collected at necropsy stained with H&E. Pulmonary arterioles appear normal in treated animals (RM2) (c, left). Treated RM2 shows mild acute bronchointerstitial pneumonia demonstrated by multifocal accumulations of inflammatory cells centered on terminal bronchioles (c, middle). Higher magnification of the boxed region showing thickening of the alveolar septae by small numbers of neutrophils, macrophages, fibrin and edema (asterisk) (c, right). Pulmonary arterioles are bounded by moderate numbers of lymphocytes indicative of perivasculitis in untreated animals (d, left). Larger numbers of inflammatory cells and fibrin also extend into alveolar spaces. Untreated RM4 shows marked coalescing bronchointerstitial pneumonia with abundant alveolar fibrin and edema (d, middle). Higher magnification of untreated RM4 showing airways filled with organizing fibrin (arrow) and moderate numbers of viable and degenerate neutrophils and macrophages (d, right). (e) Percentage of neutrophils in lung sections stained with myeloperoxidase. Two sections per lobe and six lobes per animal from three animals per group were analyzed. All values are mean ± s.d. (t-test, *P < 0.05, **P < 0.01).

  4. Transcriptional signatures.
    Figure 4: Transcriptional signatures.

    (a) Singular value decomposition–coupled multidimensional scaling (SVD-MDS) representation showing the distribution of individual samples following dimensionality reduction. This represents the similarity of the gene expression patterns of the following groups: uninfected, untreated control lung (black), MERS-CoV–infected, untreated lung (red, RM4–RM6) and MERS-CoV–infected, treated lung (blue, RM1–RM3). All lung samples were from the lower right lobe. Group means are shown as open circles, and distances from uninfected, untreated controls for MERS-CoV–infected, treated and MERS-CoV–infected, untreated macaques are shown as arrows between group means. The distance between points and groups is indicative of the differences in gene expression between the different groups. (b) Heatmap showing log10 expression ratios to untreated, MERS-CoV–infected group mean of 205 DEGs associated with IFN-α2b and ribavirin treatment, as determined by Welch's t-test (P < 0.01, fold change ≥ 1.5) and grouped by hierarchical clustering. (c) Molecular interaction network built using DEGs shown in b. Solid lines show direct molecular interactions; dashed lines show indirect molecular interactions. Red molecules are those upregulated in treated animals relative to untreated, whereas turquoise molecules are downregulated.

Accession codes

Primary accessions

Gene Expression Omnibus

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Author information

Affiliations

  1. Disease Modeling and Transmission Unit, Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, Montana, USA.

    • Darryl Falzarano,
    • Emmie de Wit,
    • Cynthia Martellaro,
    • Laura Baseler &
    • Heinz Feldmann
  2. Department of Microbiology, University of Washington, Seattle, Washington, USA.

    • Angela L Rasmussen,
    • Atsushi Okumura,
    • Arndt G Benecke &
    • Michael G Katze
  3. Rocky Mountain Veterinary Branch, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, Montana, USA.

    • Friederike Feldmann,
    • Dana P Scott &
    • Doug Brining
  4. Virus Ecology Unit, Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, Montana, USA.

    • Trenton Bushmaker &
    • Vincent J Munster
  5. Department of Comparative Pathobiology, Purdue University, West Lafayette, Indiana, USA.

    • Laura Baseler
  6. Université Pierre et Marie Curie, Centre National de la Recherche Scientifique, UMR7224, Paris, France.

    • Arndt G Benecke
  7. Washington National Primate Research Center, University of Washington, Seattle, Washington, USA.

    • Michael G Katze
  8. Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada.

    • Heinz Feldmann

Contributions

D.F., E.d.W., V.J.M. and H.F. conceived of and designed the study. D.F., E.d.W., A.L.R., F.F., A.O., D.P.S., T.B., C.M. and D.B. performed the experiments. D.F., E.d.W., A.L.R., A.O., D.P.S., L.B., A.G.B., V.J.M., M.G.K. and H.F. analyzed the data. D.F., A.L.R., M.G.K. and H.F. wrote the manuscript. This work was supported in part by the Intramural Research Program, NIAID, NIH, in addition to the NIAID Regional Centers of Excellence (U54 AI081680), Systems Virology (NIH/NIAID contract number HHSN272200800060C) and Washington National Primate Research Center (P51OD010425) to M.G.K.

Competing financial interests

The authors declare no competing financial interests.

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