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West Nile alternative open reading frame (N-NS4B/WARF4) is produced in infected West Nile Virus (WNV) cells and induces humoral response in WNV infected individuals

Giovanni Faggioni1*, Alice Pomponi1, Riccardo De Santis1, Laura Masuelli2, Andrea Ciammaruconi1, Federica Monaco3, Annapia Di Gennaro3, Laura Marzocchella4, Vittorio Sambri5, Rossella Lelli3, Giovanni Rezza6, Roberto Bei4 and Florigio Lista1

Author Affiliations

1 Histology and Molecular Biology Section, Army Medical and Veterinary Research Center Via Santo Stefano Rotondo, 4 00184, Rome, Italy

2 Department of Experimental Medicine, University of Rome “Sapienza”, Rome, Italy

3 Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Teramo, Italy

4 Department of Clinical Sciences and Translational Medicine, University of Rome “Tor Vergata”, Rome, Italy

5 Regional Reference Centre for Microbiological Emergencies (CRREM), Microbiology Unit, Azienda Ospedaliero-Universitaria di Bologna, Policlinico S. Orsola–Malpighi, Bologna, Italy

6 Department of Infectious Diseases, Istituto Superiore di Sanità, Rome, Italy

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Virology Journal 2012, 9:283  doi:10.1186/1743-422X-9-283

Published: 22 November 2012



West Nile Virus (WNV) is a flavivirus that requires an efficient humoral and cellular host response for the control of neuroinvasive infection. We previously reported the existence of six alternative open reading frame proteins in WNV genome, one of which entitled WARF4 is exclusively restricted to the lineage I of the virus. WARF4 is able to elicit antibodies in WNV infected horses; however, there was no direct experimental proof of the existence of this novel protein. The purpose of this study was to demonstrate the in vitro production of WARF4 protein following WNV infection of cultured VERO cells and its immunity in WNV infected individuals.


We produced a monoclonal antibody against WARF4 protein (MAb 3A12) which detected the novel protein in WNV lineage I-infected, cultured VERO cells while it did not react with WNV lineage II infected cells. MAb 3A12 specificity to WARF4 protein was confirmed by its reactivity to only one peptide among four analyzed that cover the full WARF4 amino acids sequence. In addition, WARF4 protein was expressed in the late phase of WNV lineage I infection. Western blotting and bioinformatics analyses strongly suggest that the protein could be translated by programmed −1 ribosomal frameshifting process. Since WARF4 is embedded in the NS4B gene, we rename this novel protein N-NS4B/WARF4. Furthermore, serological analysis shows that N-NS4B/WARF4 is able to elicit antibodies in WNV infected individuals.


N-NS4B/WARF4 is the second Alternative Reading Frame (ARF) protein that has been demonstrated to be produced following WNV infection and might represent a novel tool for a better characterization of immune response in WNV infected individuals. Further serological as well as functional studies are required to characterize the function of the N-NS4B/WARF4 protein. Since the virus might actually make an extensive use of ARFs, it appears important to investigate the novel six ARF putative proteins of WNV.

West Nile Virus; WNV; WARF4; N-NS4B/WARF4; Alternative open reading frame