Serologic assays

HTNV N-specific IgG antibodies in mice sera were determined, by enzyme-linked immunosorbent assay (ELISA) in [26,27]. 96-well microtiter plates (Costar, USA) coated with 100 μl of purified recombinant N protein of HTNV strain A9 at a concentration of 1 μg/ml as described previously. Hantaan virus glycoproteins specific IgG antibodies were evaluated by IFA using insect Sf9 cells infected with a recombinant baculovirus expressing the glycoproteins (Gn and Gc) of HTNV train A9. Titers of neutralizing antibody were also determined by microneutralization (MN) assay as previously described.

Enzyme Linked Immunospot (ELISPOT) Assay

All antibodies and reagents used in cytokine ELISPOT assays were purchased from BD/Pharmingen (San Diego, CA, USA). BD™ ELISPOT plates (BD, USA) were coated with 100 μl of anti-mouse IFN-γ Ab (5 μg/ml in Coating Buffer) at 4°C overnight. The plates were then blocked with Blocking Solution (RPMI1640) for 2 h at room temperature. 100 μl freshly isolated splenocytes (5 × 105 cells) were added into each wells and stimulated with a synthesized peptide (HTNV N protein 221-228: SVIGFLAL) at 10 μg/ml, or positive stimulators TPA (20 ng/ml) and Ionomycin (1 μg/ml). The plates were incubated for 24 h at 37°C with 5% CO2. Development and counting of cytokine ELISPOTs were performed following the manufacturer’s procedures. Spots were counted using an ELISPOT reader system (ImmunoSpot® Analyzer, USA).

Intracellular Cytokine Staining (ICS) Flow Cytometer

For the analysis of intracellular IFN-γ cytokine, freshly isolated splenocytes (5 × 106 cells) were incubated for 5 h at 37°C in RPMI containing 10% FBS and 10 μg/ml peptides (HTNV N protein 221-228: SVIGFLAL), or a positive stimulator brefeldin A (Sigma, USA) at 10 μg/ml. After being stained with FITC-conjugated anti-CD8 antibody and PE-cy5 conjugated anti-CD3 antibody (eBioscience, USA), cells were fixed with 4% paraformaldehyde in PBS for 15 min, and then were permeabilized with 0.5% saponin (Sigma, USA) in PBS for 10 min. Finally, cells were stained with PE-conjugated anti-mouse IFN-γ McAb. All the procedures of antibody staining were performed at room temperature for 15 min. Cell samples were then analyzed with an Epics-MCL Cytometer (Beckman Coulter, USA), and the data were collected with EXPO32 ADC XL 4 Color Software.

Statistical analysis

Statistical significance of the data was determined by using Student’s t test or ANOVA of the SPSS 10.0 software. The antibody titers were log10 transformed to get a normal distribution before statistical analysis. A P value of < 0.05 was considered significant. Ethical approval

According to the medical research regulation of Ministry of Health, China, this study was approved by the ethics committee of China CDC, which uses international guidelines to ensure confidentiality, anonymity, and informed consent. Informed consent was obtained from all study participants.