Use of Saccharomyces cerevisiae -Expressed Recombinant Nucleocapsid Protein To Detect Hantaan Virus-Specific Immunoglobulin G (IgG) and IgM in Oral Fluid

Abstract

Hantaan virus (HTNV) is the prototype species of the genus Hantavirus and has remained the epidemiologically most important species in the genus until now. Hantaviruses form a separate genus within the family Bunyaviridae, forming a group of closely related negative-stranded RNA viruses (4,17,25,29). Hantaviruses are the causative agents of hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome. HFRS occurs mainly in Europe and Asia and is associated with HTNV, Seoul virus (SEOV), Dobrava virus (DOBV), and Puumala virus (PUUV) infections (34). Usually, hantavirus pulmonary syndrome is found in the New World, where most of the cases are caused by the Sin Nombre virus (SNV) and Andes virus (ANDV) serotypes (8,11,15). Hantaviruses are transmitted to humans through the aerosol excreta of infected small mammals, mainly wild rodents (17). In China, HTNV together with SEOV accounts for at least 100,000 cases of hantavirus-associated HFRS each year. Approximately 150,000 HFRS cases occur annually worldwide. HTNV infection is characterized by fever, renal dysfunction, and in some cases, hemorrhagic manifestations (9,10,12,13,15).Hantaviruses have a tripartite genome that encodes a nucleocapsid protein (N protein), two envelope glycoproteins, and a viral RNA polymerase (30). The viral N protein elicits a strong humoral immune response in infected patients and immunized animals and has been extensively used to produce reagents for the diagnosis of hantavirus infections (1, 14, 21). Comparison of the amino acid sequences of five amino-terminal immunogenic regions of the N proteins of the HTNV, SEOV, and DOBV hantaviruses shows that they have high degrees of homology. Rabbit polyclonal sera produced against cell culture-grown virus and recombinant viral proteins were tested for their responses to five hantavirus antigens. Two types of antibody responses were generated; hence, two groups of sera could be distinguished, with HTNV, SEOV, and DOBV be attributed to the first group and SNV and PUUV to the second group (3). Schmidt and coworkers (31) investigated the cross-reactivity of rabbit sera raised against the N proteins of SEOV and other hantaviruses. The N-protein-specific antibodies induced by natural infection in human and experimental infections and immunizations have been found to be highly cross-reactive among the different hantaviruses (16,26,31). The N-protein-specific antibody titers in SEOV N-protein-immunized rabbits were only slightly lower than those in rabbits immunized with the N protein of the closely related HTNV and much lower than those in rabbits immunized with the N proteins of PUUV, SNV, and ANDV. Similarly, the anti-

Phylogenetic analysis of Puumala virus subtype Bavaria, characterization and diagnostic use of its recombinant nucleocapsid protein

“…In line with data obtained for yeast-expressed HTNV antigen [83,84], we found a very similar sensitivity and specificity of the PUUV-Bawa antigen-based indirect ELISA (99 and 100%) and mAb-capture ELISA (95 and 100%) for the serum panel from Germany. A comparison of the results of the validation of the indirect PUUV-Vra ELISA and the PUUV-Bawa mAb-capture ELISA for all investigated sera from Finland showed a lower sensitivity for the capture ELISA (89%) than for the indirect ELISA (100%), but a higher specificity (100 and 95%, respectively).…”

Generation of Recombinant Schmallenberg Virus Nucleocapsid Protein in Yeast and Development of Virus-Specific Monoclonal Antibodies

“…The reactivity of yeast-expressed SBV N protein with SBV IgG-positive cow sera is in line with previous studies that demonstrated the usefulness of recombinant viral N proteins expressed in different heterologous systems for serological diagnosis of bunyaviruses infection both by western blot and ELISA [17–21]. The N protein of bunyaviruses is the most abundant viral antigen present in the virion and in the infected cells, thus making it an excellent target for serology as well as epidemiological studies of viral infections [16–18].…”

“…The N protein of bunyaviruses is the most abundant viral antigen present in the virion and in the infected cells, thus making it an excellent target for serology as well as epidemiological studies of viral infections [ 16 – 18 ]. Recombinant N protein of hantaviruses, generated in E. coli , insect cells, or yeast, has been widely used for detection of hantavirus-specific antibodies in human sera and oral fluid [ 17 – 21 ]. SBV N protein was expressed recently in E. coli [ 11 , 32 ] and used for the development of a commercial diagnostic kit for serologic diagnosis of SBV infection [ 11 ].…”

Seroepidemiological study in a Puumala virus outbreak area in South-East Germany

“…[23,24,29,30] and indirect ELISAs for SNV, PUUV, HTNV and ANDV using E. coliexpressed N antigens [8,21,34]. The seroepidemiological study in four counties of two administrative districts of Lower Bavaria revealed an overall prevalence of 6.7% with a range of 0% in Schöfweg (district Freyung-Grafenau), 4.1% in Geiersthal (district Regen), 5.9% in Kirchberg (district Regen) and 11.9% in Spiegelau (district Freyung-Grafenau).…”