All authors read and approved the final manuscript. Notes Ethics approval and consent to participate This work was approved by the ethics committee of China CDC. viruses made up of three single-stranded RNA genome segments designated as small (S), medium (M) and large (L); they encode nucleocapsid protein (N), envelope glycoproteins (Gn and Gc) and RNA-dependent RNA polymerase, respectively [2, 3]. Among the viral proteins, nucleocapsid protein possesses an immunodominant antigen, and the antigenicitiy of N protein is conserved compared with that of envelope glycoproteins [4, 5]. Gn and Gc form oligomers on the surface of the virion and are the targets of neutralizing antibodies [6C8]. Hantavirus causes two human diseases: hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus pulmonary syndrome (HPS) in the Americas. At least four hantaviruses cause HFRS: Hantaan, Seoul, Puumala, and Dobrava viruses caused most of HFRS cases in Eurasia [9, 10]. Hantaan computer virus (HTNV) and Seoul computer virus (SEOV) are major causative brokers of HFRS in China [11], During the last decade, about 10,000 cases of HFRS were registered annually in China [12]. In general, hantaviruses are host-restricted that Hantaan computer virus isolates are carried by and Seoul computer virus isolates by [1]. The plaque reduction neutralization test (PRNT), is usually laborious and time-consuming (takes about 2?~?3?weeks), and is unsuitable for high-throughput testing [13C15]. Therefore, option methods to PRNT are needed. Microneutralization test (MNT) has been developed for viruses such as influenza computer virus, Puumala computer virus, etc. [16C22]. By using 96-well microplates in combination with enzyme immunoassay, MNT is simple, rapid, and adaptable to high-throughput formats. Pseudotyped reporter viruses made up of the envelope glycoprotein of one virus and the core and genome of vector computer virus such as vesicular stomatitis computer virus (VSV), murine leukemia computer virus (MuLV) or lentivirus have been developed for many other viruses [23C27]. Since pseudoparticle is unable to produce infectious progeny viruses unless AS-252424 the envelope proteins are provided in pseudoparticle neutralization test (PPNT) is usually a safe alternative to neutralization test using live viruses. Pseudoparticles bearing glycoproteins of hantaviruses have also been developed and used in PPNT AS-252424 [28C33] for hantaviruses. Here, we compared the MNT and PPNT data with those obtained with PRNT using 44 convalescent sera from laboratory confirmed patients of HFRS and 30 sera unfavorable for hantavirus contamination. Moreover, the effective expressions of glycoproteins of HTNV and SEOV in 293T cells enable us to develop a method of immunofluorescence assay based on viral glycoproteins (IFA-GP) to detect antibody titres against recombinant glycoproteins of the two viruses. The IFA-GP titres may correlate with the neutralizing antibody titres obtained by PRNT, thus IFA-GP has the possibility to be a simpler alternative to PRNT. Here, results obtained with IFA-GP were also compared with that obtained with PRNT using the same panel of sera mentioned above. Methods Cells, viruses, antibody Vero-E6 cells (ATCC, C1008 CRL1586) were propagated in growth Rabbit Polyclonal to NCAPG medium (Eagles MEM supplemented with 10% heat-inactivated fetal bovine serum [FBS], 2?mM L-glutamine, 100?U/ml Penicillin, 100?g/ml Streptomycin, and 1.5?g/L Sodium Bicarbonate solution). HEK 293T human embryo kidney cells (ATCC, “type”:”entrez-protein”,”attrs”:”text”:”CRL11268″,”term_id”:”903511506″CRL11268) and Huh-7.5 human hepatocellular carcinoma cells [34] were propagated in Dulbeccos Modified Eagle Medium [DMEM] containing 10% heat-inactivated FBS, 100?U/ml Penicillin, 100?g/ml Streptomycin. HTNV strain 84FLi and SEOV strain L99 maintained in our laboratory were propagated on Vero E6 cells. The mouse monoclonal antibodies (mAbs) L13F3 AS-252424 directed against N AS-252424 protein of SEOV and HTNV were generated in our laboratory [35]. Mouse mAbs 8B6 directed against hantavirus Gn glycoprotein [6] and human recombinant mAbs Y5 directed against hantavirus Gc glycoprotein [36] were stored in our laboratory. Serum samples A panel of 74 human sera was used in this study, including 44 convalescent sera from laboratory confirmed patients of HFRS in China, 15 sera from healthy individuals and 15 AS-252424 sera from patients of dengue.