Phage Displayed Peptides to Avian H5N1 Virus Distinguished the Virus from Other Viruses

PLOS ONE, Aug 2011

The purpose of the current study was to identify potential ligands and develop a novel diagnostic test to highly pathogenic avian influenza A virus (HPAI), subtype H5N1 viruses using phage display technology. The H5N1 viruses were used as an immobilized target in a biopanning process using a 12-mer phage display random peptide library. After five rounds of panning, three phages expressing peptides HAWDPIPARDPF, AAWHLIVALAPN or ATSHLHVRLPSK had a specific binding activity to H5N1 viruses were isolated. Putative binding motifs to H5N1 viruses were identified by DNA sequencing. In terms of the minimum quantity of viruses, the phage-based ELISA was better than antiserum-based ELISA and a manual, semi-quantitative endpoint RT-PCR for detecting H5N1 viruses. More importantly, the selected phages bearing the specific peptides to H5N1 viruses were capable of differentiating this virus from other avian viruses in enzyme-linked immunosorbent assays.

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Phage Displayed Peptides to Avian H5N1 Virus Distinguished the Virus from Other Viruses

Citation: Wu D, Li G, Qin C, Ren X ( Phage Displayed Peptides to Avian H5N1 Virus Distinguished the Virus from Other Viruses Dan Wu 0 Guangxing Li 0 Chengfeng Qin 0 Xiaofeng Ren 0 Paul Digard, University of Cambridge, United Kingdom 0 1 Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University , Harbin , China , 2 Department of Basic Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University , Harbin , China , 3 State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology , Beijing , China The purpose of the current study was to identify potential ligands and develop a novel diagnostic test to highly pathogenic avian influenza A virus (HPAI), subtype H5N1 viruses using phage display technology. The H5N1 viruses were used as an immobilized target in a biopanning process using a 12-mer phage display random peptide library. After five rounds of panning, three phages expressing peptides HAWDPIPARDPF, AAWHLIVALAPN or ATSHLHVRLPSK had a specific binding activity to H5N1 viruses were isolated. Putative binding motifs to H5N1 viruses were identified by DNA sequencing. In terms of the minimum quantity of viruses, the phage-based ELISA was better than antiserum-based ELISA and a manual, semiquantitative endpoint RT-PCR for detecting H5N1 viruses. More importantly, the selected phages bearing the specific peptides to H5N1 viruses were capable of differentiating this virus from other avian viruses in enzyme-linked immunosorbent assays. - Funding: This work was supported, in part, by the National 973 Plan of China (2010CB534002). We also acknowledge National Natural Science Foundation of China (30972195), the Program for New Century Excellent Talents in Heilongjiang Provincial University (1155-NCET-005) and Heilongjiang Provincial Science and Technology Department, China (ZJN0702-01). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Since the first evidence regarding direct transmission of highly pathogenic avian influenza A virus (HPAI), subtype H5N1 from poultry to human in 1997 and resulted in the death of 6 of the 18 infected individuals [13]. The HPAI H5N1 has become one of the most important public health concerns worldwide. At present, the virus has spread to many countries in Europe, Asia and Africa [4]. In 2009, an identified fatal influenza (H5N1) infection in a human was reported on January 17, 2009 [5]. Increased geographical distribution and continued evolution of H5N1 viruses as well as an immunologically nave human population has maintained the pandemic potential of these viruses [68]. In addition to vaccination and administration of antiviral drugs against H5N1 viruses, development of effective detection approaches is required to manage and control the deadly disease. Phage display is a recently developed technology and phage random peptide library consists of a pool of billions of heterologous peptides that can be produced by the fusion of random nucleic acid sequences to the N terminus of one of the capsid protein genes of a filamentous bacteriophage [9]. Phage display peptide library is a powerful tool to identify specific ligands of a target protein by a biopanning process. This technology has been applied successfully in numerous aspects, including antibody engineering [10], peptide and protein drug discovery and manufacture [11], diagnostic analysis [12] and vaccine development [13]. Herein we identified three phage clones that specifically binding to the HAPI H5N1 viruses using a 12-mer random phage library. The binding peptides of the phages were sequenced. More importantly, these identified phages were able to distinguish HAPI H5N1 from other avian viruses. Materials and Methods Cell and virus Madin-Darby canine kidney (MDCK) cells (ATCC, Manassas, VA) were grown in Dulbeccos MEM with 1 mM L-glutamine and 10% fetal bovine serum at 37uC and 5% CO2 in air. HPAI H5N1 strain A/goose/Jilin/hb/2003 were propagated in the MDCK cells in the absence of serum and purified by differential centrifugation conventionally. The concentration of the purified viruses diluted in PBS was measured by Thermo Scientific NANODROP 2000 Spectrophotometer ((NanoDrop Technologies, Thermo Fisher Scientific, Wilmington, DE) and calculated by the molar absorbance coefficient A260/A280 according to the manufacturers instructions. Biopanning and enrichment analysis Phage display was done according to the manufacturers instructions (New England Biolabs) with minor modifications. For the first round of panning, 96-well plates were coated with the H5N1 viruses at a concentration of 14 mg/well in 0.1 M NaHCO3 (pH 8.6) buffer overnight at 4uC. The next day, the plates were blocked for 1 h at 4uC (...truncated)


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Dan Wu, Guangxing Li, Chengfeng Qin, Xiaofeng Ren. Phage Displayed Peptides to Avian H5N1 Virus Distinguished the Virus from Other Viruses, PLOS ONE, 2011, Volume 6, Issue 8, DOI: 10.1371/journal.pone.0023058