Identification Of One Peptide With Infectious Bronchitis Virus (IBV) Neutralizing Activity In Vitro And Cloning Of The S Gene Of IBV H52 Strain

Coronaviruses, comprising a genus of Coronaviridae family, are large, enveloped viruses with single stranded, positive-sense RNA genomes. Avian infectious bronchitis virus (IBV), a prototype of coronaviruses, belongs to group III coronaviruses. IBV is the pathogen of chicken infectious bronchitis (IB), which is an acute, highly contagious respiratory disease in young chickens. More than 29 IBV serotypes have been identified worldwide and there is an increasing number of new serotypic variants reported. The specific interaction between the spike (S) protein and cellular receptor(s) is directly involved in host specificity and tissue or cell tropism of IBV. Cloning of S gene is important for studying IBV.Phage display is a advanced molecular technology that allows the presentation of large peptides or protein on the surface of filamentous phage by fusing sequence of a target gene with the phage capsid gene. A crucial advantage of this technology is relatively independent three dimensional structure and biological activity of the displayed peptide or protein, which allows directly selection and study of functional domains of binding parteners such as antigen-antibody or receptor-ligand.This study utilized purified IBV H52 strain to screen specific virus-binding peptides from Ph. D.-12TM phage display peptide library.Binding efficiency of the selected phages was confirmed by ELISA. Several recovered phages were selected to be sequenced after the 3rd and 4th round of panning against purified virions. The sequences were aligned and analyzed and a common histidine-rich motif "HHXHXS" with some flexibility in the residue number between "H" and "S" was identified. Phages containing the motif can specifically inhibit IBV haemagglutination to avian red cells and neutralize the infectivity of IBV in a human cell line HeLa, but could not block the reaction between IBV and its antiserum. In contrast, these IBV binding phages did not inhibit haemagglutination of other avian viruses including Newcastle disease virus (NDV) and avian influenza virus (AIV) H9N2. Phages displaying unrelated peptides did not have this HI and neutralization activity. One linear peptide "N- GSH HRH VHS PFV-C" from the positive phages was synthesized and found to inhibited IBV infection of HeLa cells with the highest neutralization titer compared to all phages. This study may contribute to the development of antiviral therapeutics for IBV and definition of the neutralization epitopes.The full length of S gene of IBV was cloned using the allantoic fluid infected with IBV and characterized. The results showed that the cloned sequence shares 99% identity with M41 strain (GenBank accession number: M21883.1) and shares 97% identity with strain Beaudette CK (GenBank accession number: AJ311317) respectively. Other traits were determined with bioinformatics tools.