Genotyping and preparation of the recombinant nucleocapsid protein antigen of hantavirus.
OBJECTIVE
To identify new recombinant antigens with potential for diagnosis of hemorrhagic fever with renal syndrome (HFRS) and establish reverse transcription-polymerase chain reaction-restriction fragments length polymorphism (RT-PCR-RFLP) for genotyping of hantavirus.


METHODS
One group of primers was used to clone the full-length S genome segment and the partial S genome segment of the N-terminal. The two cloned genes were both fusionally expressed and non-fusionally expressed in the T7 system. The other group of primers was used to establish a RT-PCR method to detect RNAs in 37 virus isolates, 2 positive standard virus strains of hantavirus and 5 negative controls. The method for typing RFLP was set up by digesting the PCR products of 20 virus isolates with Ras I and Hind III.


RESULTS
The non-fusionally expressed products with a working concentration of 1:10,000 by chapping enzyme-linked immunosorbent assay (cELISA), presented good biological activity though yields were lower than that of the fusionally expressed products. The specific component of the hantavirus genome (299 bp or 577 bp) was seen in all viral samples. The genotyping of hantavirus showed that 9 out of the total were Hantann (HTN) viruses, 8 were seoul (SEO) viruses and 3 were not determined.


CONCLUSIONS
The good working titrer of expressed recombinant antigen showed that it has the potential to replace the natural antigen for detecting hantavirus antibodies. On comparison with cELISA, the detection rates for these two methods were 100% and 84.6%, and the coincidence rate was 84.6%. The former had a 15.4% higher sensitivity than the latter. The typing efficiency of RT-PCR-RFLP and sero-typing method was 85% (17/20) and 55% (11/20), respectively, showing that the former was 30% higher than the latter, while their results were highly consistent.
Tang
J
J
Research Institute for Military Medical Sciences, Nanjing 210002, China.
Cao
M
M
Tang
T
T
Wang
C
C
Wei
C
C
Lei
W
W
D000914:Antibodies, Viral / Q000097:blood; D004797:Enzyme-Linked Immunosorbent Assay; D005838:Genotype; D006476:Orthohantavirus / Q000145:classification / Q000235:genetics / Q000276:immunology*; D018778:Hantavirus Infections / Q000175:diagnosis*; D006801:Humans; D019590:Nucleocapsid Proteins / Q000276:immunology*; D012150:Polymorphism, Restriction Fragment Length; D011994:Recombinant Proteins / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D000914:Antibodies, Viral; D019590:Nucleocapsid Proteins; D011994:Recombinant Proteins