title: Synthesis of human parainfluenza virus 4 nucleocapsid-like particles in yeast and their use for detection of virus-specific antibodies in human serum.
abstract: The aim of this study was to produce human parainfluenza virus type 4 (HPIV4) nucleocapsid (N) protein in yeast Saccharomyces cerevisiae expression system, to explore its structural and antigenic properties and to evaluate its applicability in serology. The use of an optimized gene encoding HPIV4 N protein amino acid (aa) sequence GenBank AGU90031.1 allowed high yield of recombinant N protein forming nucleocapsid-like particles (NLPs) in yeast. A substitution L332D disrupted self-assembly of NLPs, confirming the role of this position in the N proteins of Paramyxovirinae. Three monoclonal antibodies (MAbs) were generated against the NLP-forming HPIV4 N protein. They recognised HPIV4-infected cells, demonstrating the antigenic similarity between the recombinant and virus-derived N proteins. HPIV4 N protein was used as a coating antigen in an indirect IgG ELISA with serum specimens of 154 patients with respiratory tract infection. The same serum specimens were tested with previously generated N protein of a closely related HPIV2, another representative of genus Rubulavirus. Competitive ELISA was developed using related yeast-produced viral antigens to deplete the cross-reactive serum antibodies. In the ELISA either without or with competition using heterologous HPIV (2 or 4) N or mumps virus N proteins, the seroprevalence of HPIV4 N-specific IgG was, respectively, 46.8, 39.6 and 40.3% and the seroprevalence of HPIV2 N-specific IgG-47.4, 39.0 and 37.7%. In conclusion, yeast-produced HPIV4 N protein shares structural and antigenic properties of the native virus nucleocapsids. Yeast-produced HPIV4 and HPIV2 NLPs are prospective tools in serology.
authors.affiliation: Institute of Biotechnology, Vilnius University, Saulėtekio 7, LT-10257, Vilnius, Lithuania. aiste.bulavaite@bti.vu.lt.
authors.forename: Aistė
authors.identifier: http://orcid.org/0000-0002-4219-3985
authors.initials: A
authors.lastname: Bulavaitė
authors.affiliation: Institute of Biotechnology, Vilnius University, Saulėtekio 7, LT-10257, Vilnius, Lithuania.
authors.forename: Rita
authors.initials: R
authors.lastname: Lasickienė
authors.affiliation: Institute of Biotechnology, Vilnius University, Saulėtekio 7, LT-10257, Vilnius, Lithuania.
authors.forename: Paulius Lukas
authors.initials: PL
authors.lastname: Tamošiūnas
authors.affiliation: Institute of Biotechnology, Vilnius University, Saulėtekio 7, LT-10257, Vilnius, Lithuania.
authors.forename: Martynas
authors.initials: M
authors.lastname: Simanavičius
authors.affiliation: Institute of Biotechnology, Vilnius University, Saulėtekio 7, LT-10257, Vilnius, Lithuania.
authors.forename: Kęstutis
authors.initials: K
authors.lastname: Sasnauskas
authors.affiliation: Institute of Biotechnology, Vilnius University, Saulėtekio 7, LT-10257, Vilnius, Lithuania.
authors.forename: Aurelija
authors.initials: A
authors.lastname: Žvirblienė
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000097:blood*; D000956:Antigens, Viral / Q000276:immunology; D002648:Child; D002675:Child, Preschool; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood*; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D008875:Middle Aged; D019251:Nucleocapsid; D019590:Nucleocapsid Proteins / Q000097:blood / Q000235:genetics / Q000276:immunology*; D010223:Parainfluenza Virus 2, Human / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D029482:Parainfluenza Virus 4, Human / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D011446:Prospective Studies; D011994:Recombinant Proteins / Q000276:immunology; D012141:Respiratory Tract Infections / Q000276:immunology* / Q000821:virology; D019351:Rubulavirus Infections / Q000276:immunology*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D016036:Seroepidemiologic Studies; D055815:Young Adult
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral; D007074:Immunoglobulin G; D019590:Nucleocapsid Proteins; D011994:Recombinant Proteins
keywords: ELISA; Human parainfluenza virus 2; Human parainfluenza virus 4; Nucleocapsid-like particles; Serology; Yeast-produced N protein