title: Generation of recombinant metapneumovirus nucleocapsid protein as nucleocapsid-like particles and development of virus-specific monoclonal antibodies.
abstract: Human metapneumovirus (hMPV) is a member of the Pneumovirinea subfamily within the Paramyxoviridea family. Since its discovery in 2001, hMPV has been isolated in several continents, which suggests its prevalence worldwide. hMPV resembles human respiratory syncytial virus with regard to disease symptoms and its ability to infect and cause disease in young infants as well as individuals of all ages. The aim of the current study was to construct an efficient high-level yeast expression system for the generation of hMPV nucleocapsid (N) protein and to develop monoclonal antibodies (MAbs) suitable for hMPV detection. The genome of hMPV was isolated from oral fluid of an infected patient by using specific primers and reverse transcriptase polymerase chain reaction (RT-PCR). DNA sequence corresponding to the N protein gene was inserted into yeast expression vector under inducible GAL7 promoter. SDS-PAGE analysis of crude lysates of yeast Saccharomyces cerevisiae harbouring recombinant plasmid revealed the presence of a protein band of approximately 43 kDa corresponding to the molecular weight of hMPV N protein. Electron microscopy analysis of purified N protein revealed nucleocapsid-like structures with typical herring-bone morphology: rods of 20 nm diameter with repeated serration along the edges and central core of 5 nm. Recombinant hMPV N protein was reactive with human serum specimens collected from patients with confirmed hMPV infection. After immunization of mice with recombinant hMPV N protein, a panel of MAbs was generated. The specificity of newly generated MAbs was proven by immunofluorescence analysis of hMPV-infected cells. Epitope mapping using truncated variants of hMPV N revealed localization of linear MAb epitopes at the N-terminus of hMPV N protein, between amino acid residues 1 and 90. The MAbs directed against conformational epitopes did not recognize hMPV N protein variants containing either N- or C-terminal truncations. The reactivity of recombinant hMPV N protein with hMPV-positive serum specimens and the ability of MAbs to recognize virus-infected cells confirms the antigenic similarity between yeast-expressed hMPV N protein and native viral nucleocapsids. In conclusion, recombinant hMPV N protein and hMPV-specific MAbs provide new diagnostic reagents for hMPV infection.
authors.affiliation: Institute of Biotechnology, Vilnius University, V.Graiciuno 8, 02241 Vilnius, Lithuania. prasa@ibt.lt
authors.forename: Rasa
authors.initials: R
authors.lastname: Petraitytė-Burneikienė
authors.forename: Kristina
authors.initials: K
authors.lastname: Nalivaiko
authors.forename: Rita
authors.initials: R
authors.lastname: Lasickienė
authors.forename: Regina
authors.initials: R
authors.lastname: Firantienė
authors.forename: Regina
authors.initials: R
authors.lastname: Emužytė
authors.forename: Kęstutis
authors.initials: K
authors.lastname: Sasnauskas
authors.forename: Aurelija
authors.initials: A
authors.lastname: Zvirblienė
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000914:Antibodies, Viral / Q000276:immunology*; D002460:Cell Line; D002648:Child; D018604:Epitope Mapping; D005260:Female; D006801:Humans; D008297:Male; D029121:Metapneumovirus / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D018184:Paramyxoviridae Infections / Q000276:immunology* / Q000821:virology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D019590:Nucleocapsid Proteins; D011994:Recombinant Proteins