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Technical Briefs |
1
School of Chemistry and Material Science, Shaanxi Normal University, 710062, Xi’an, China;
2
Department of Infectious Disease, Tangdu Hospital,
3
Cell Engineering Research Centre, Fourth Military Medical University, 710032, Xi’an, China
aauthor for correspondence: fax 86-29-3293906, e-mail bhj{at}pub.xaonline.com
Hantavirus (HV) is the causative agent of a severe type of hemorrhagic fever with renal syndrome (HFRS), with an annual incidence in China of 50 000–100 000 cases. Hantaan virus (HTNV) 76–118 is the prototype strain of the HV genus and the hantaan serotype (1). HV has a single-stranded, negative-sense tripartite RNA genome, the segments of which are designated large, medium, and small. The RNA genome encodes the viral RNA polymerase, envelope glycoproteins (G1, G2), and nucleocapsid protein (NP) (2). A serologic investigation (3) showed that HTNV NP has strong antigenicity and immunogenicity, and the antibodies against NP in patients with HFRS not only appear early, but also have high titer. Moreover, NP contains the major antigen that can cross-react with the immunized sera of many serologically and genetically distinct groups of HV (4). To screen the epitopes of NP with monoclonal antibodies against HTNV, a library of HTNV small gene-peptide fragments on phages was constructed. Results showed that a linear epitope consisting of amino acids 1–86 (aa 1–86) within the NP reading frame was determined and its core sequence consists of aa 15–66, which is a major and cross-reactive antigenic epitope (5).
Earlier serologic assays for the detection of HTNV-specific immune responses were based on virus propagated in cell culture (6). The hazardous nature of HTNV, its slow replication, and low yield in cell culture have prompted expression of recombinant HTNV NP for use as an antigen in ELISA (7). However, the expression and purification of recombinant HTNV NP is not straightforward for either the full-length or partial recombinant HTNV NP (8). It is necessary to establish a novel specific serologic diagnosis of HTNV infection.
We developed an ELISA-based method for the detection of HFRS, using a synthetic peptide antigen. Synthetic peptides that mimic specific epitopes of infectious viral proteins have been used in diagnostic systems for various diseases and for the molecular design of vaccines (9)(10). Synthetic peptides offer the advantage of eliminating nonspecific reactions resulting from the cross-reactions of antibodies in the specimen with host-cell-derived viruses or recombinant products derived from Escherichia coli (11).
For computer-assisted epitope prediction, we used the software Antheprot V4.3c (http://pbil@ibcp.fr) and predicted hydrophobicity and antigenicity, together with predictions of 
, ß, turn, and coil conformation probabilities. This indicated that the most probable antigenic site of HTNV NP is between aa 17 and aa 66 with the NH2 terminus reading frame. Then the peptide (aa 17–66) of HTNV NP was synthesized by solid-phase peptide synthesis. The crude peptide was first purified by a G-25 column. The preparation and analysis of the peptide was then performed with reversed-phase HPLC. The results from amino acid analysis were as follows: Thr1.9Ser1.03 Ala3.82Asp5.03Glu5.02Gly1Ile2.86Lys6.86Leu5.03Asn1.02Pro1.03 Gln5Arg5.04Val4Tyr0.9.
Micro-ELISA plates were coated with the synthetic peptide in 0.02 mol/L Tris-HCl buffer (pH 8.0) for 12 h at 37°C. The peptides were used in a free form. Free binding sites were saturated by incubation with 30 g/L bovine serum albumin in phosphate-buffered saline (PBS). After 1 h of incubation at 37 °C, the wells were washed four times with PBS-Tween 20 (PBST). Sera were diluted 1:100 (1 volume of serum + 99 volumes of PBST) in PBST with normal goat serum (200 mL/L) containing Triton X-100 (5 mL/L). The mixture was incubated for 1 h at 37 °C. For IgM detection, the sera were first treated with 10 µL of goat antibodies to human IgG to remove the IgG. After washing, horseradish peroxidase (HRP)-conjugated goat antibodies to human IgG or IgM diluted 1000-fold in PBST containing 10 g/L bovine serum albumin was added for 1 h. The wells were washed four times with PBST, and the bound HRP label was detected with 3,3',5,5'-tetramethylbenzidine as the substrate for 30 min in the dark, after which time the color reaction was stopped by the addition of 2 mol/L H2SO4. The cutoff for positive was defined as the mean absorbance (+3 SD) at 492 nm for 81 control sera; the cutoff value was 0.070.
Molecular characterization of the IgM and IgG immune responses against HRFS was performed by analysis of serum samples. The synthetic peptide was applied as an antigen to test the specific antibody (IgM, IgG) by ELISA. Forty-six sera from HFRS patients, 9 sera from HBV patients, and 81 sera from healthy people were analyzed. The samples from healthy volunteers were analyzed in the same run as the HFRS samples. The analyst knew which samples were from which group (HRFS, HBV, and healthy volunteers). In this preliminary study, the sera of the HBV patients and healthy individuals were negative for anti-aa 17–66 IgM, and 4 sera of healthy people were positive for anti-aa 17–66 IgG. Among HFRS patients, 43 of 46 sera were positive for anti-aa 17–66 IgM (Fig. 1A
), and 42 of 46 sera were positive for anti-aa 17–66 IgG (Fig. 1B
). Future clinical studies should be performed to better establish the sensitivity and specificity of this assay.
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Acknowledgments
This project was supported by grants from the Natural Science Foundation of Shaanxi province, China (Project No. 691049).
References
The following articles in journals at HighWire Press have cited this article:
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  R. Petraityte, L. Jin, R. Hunjan, A. Razanskiene, A. Zvirbliene, and K. Sasnauskas Use of Saccharomyces cerevisiae-Expressed Recombinant Nucleocapsid Protein To Detect Hantaan Virus-Specific Immunoglobulin G (IgG) and IgM in Oral Fluid Clin. Vaccine Immunol., December 1, 2007; 14(12): 1603 - 1608. [Abstract] [Full Text] [PDF]
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H. Meisel, A. Wolbert, A. Razanskiene, A. Marg, A. Kazaks, K. Sasnauskas, G. Pauli, R. Ulrich, and D. H. Kruger Development of Novel Immunoglobulin G (IgG), IgA, and IgM Enzyme Immunoassays Based on Recombinant Puumala and Dobrava Hantavirus Nucleocapsid Proteins Clin. Vaccine Immunol., December 1, 2006; 13(12): 1349 - 1357. [Abstract] [Full Text] [PDF]
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 W. Muranyi, U. Bahr, M. Zeier, and F. J. van der Woude Hantavirus Infection J. Am. Soc. Nephrol., December 1, 2005; 16(12): 3669 - 3679. [Full Text] [PDF]
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