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- Elgh, Fredrik, 1957-, et al.
(författare)
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Serological diagnosis of hantavirus infections by an enzyme-linked immunosorbent assay based on detection of immunoglobulin G and M responses to recombinant nucleocapsid proteins of five viral serotypes
- 1997
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Ingår i: Journal of clinical microbiology. - : American Society for Microbiology. - 0095-1137 .- 1098-660X. ; 35:5, s. 1122-1130
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Tidskriftsartikel (refereegranskat)abstract
- Worldwide, hantaviruses cause more than 100,000 human infections annually. Rapid and accurate methods are important both in monitoring acute infections and for epidemiological studies. We and others have shown that the amino termini of hantavirus nucleocapsid proteins (Ns) are sensitive tools for the detection of specific antibodies in hantavirus disease. Accordingly, we expressed truncated Ns (amino acids 1 to 117) in Escherichia coli from the five hantaviruses known to be pathogenic to man; Hantaan (HTN), Seoul (SEO), Dobrava (DOB), Sin Nombre (SN), and Puumala (PUU) viruses. In order to obtain pure antigens for use in an enzyme-linked immunosorbent assay (ELISA), the recombinant proteins were purified by polyhistidine-metal chelate affinity chromatography. Polyclonal animal antisera and a panel of serum specimens from hantavirus-infected individuals from Scandinavia, Slovenia, Russia, Korea, China, and the United States were used to evaluate the usefulness of the method. With both human and animal sera, it was possible to designate the antibody response into two groups: those with HTN, SEO, and DOB virus reactivity on the one hand and those with SN and PUU virus reactivity on the other. In sera from Scandinavia, European Russia, and the United States, the antibody response was directed mainly to the PUU and SN virus group. The sera from Asia reacted almost exclusively with the HTN, SEO, and DOB types of viruses. This was true for both the immunoglobulin M (IgM) and IgG antibody responses, indicating that this type of discrimination can be done during the acute phase of hantavirus infections. Both the HTN, SEO, and DOB virus and the PUU and SN virus types of antibody response patterns were found in patients from the Balkan region (Solvenia).
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- HORLING, J, et al.
(författare)
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Detection and subsequent sequencing of Puumala virus from human specimens by PCR
- 1995
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Ingår i: Journal of clinical microbiology. - : American Society for Microbiology. - 0095-1137 .- 1098-660X. ; 33:2, s. 277-282
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Tidskriftsartikel (refereegranskat)abstract
- A sensitive method based on PCR was developed for the detection of Puumala virus (PUU) in human samples. The assay was found to be specific for PUU-like strains and distinguished between these and hantaviruses of other serotypes. The detection limit was found to be 10(-5) focus-forming units. Clinical samples were collected from patients with nephropathia epidemica in Sweden and western Russia. Five whole blood samples collected from patients in Russia with the acute phase of disease were found to be positive by the PCR. All samples were negative for PUU antigen when examined by enzyme-linked immunosorbent assay. Virus isolation on Vero E6 cells from several of the acute-phase samples, including the 5 PCR-positive samples, was not successful. The amplified samples were subjected to direct nucleic acid sequencing for confirmation of identity. The sequences differed from each other and were closely related to the Russian bank vole isolate CG-1820, thereby indicating the origin of nephropathia epidemica. The PCR was used for amplification and subsequent nucleotide sequencing of eight PUU-like isolates with different geographic origins. The Swedish strains were more closely related to the Finnish PUU prototype strain, Sotkamo, than to the Russian isolates. Interestingly, a Belgian isolate, CG-13891, differed markedly from all other PUU strains.
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| 6. |
- Plyusnin, A, et al.
(författare)
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Puumala hantavirus genome in patients with nephropathia epidemica: correlation of PCR positivity with HLA haplotype and link to viral sequences in local rodents
- 1997
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Ingår i: Journal of clinical microbiology. - : American Society for Microbiology. - 0095-1137 .- 1098-660X. ; 35:5, s. 1090-1096
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Tidskriftsartikel (refereegranskat)abstract
- Reverse transcription-PCR was used to analyze specimens from 20 Finnish nephropathia epidemica (NE) patients hospitalized during the period from October 1994 to January 1995. Blood and/or urine sediment specimens from seven patients were found to be positive for the genome sequences of Puumala hantavirus (PUU). PCR positivity of the specimens from the patients correlated well with the HLA-DRB1*0301 and HLA B8 alleles, which previously were shown to associate with severe courses of NE. Genetic analysis of the partial M-and/or S-segment sequences obtained from three severely ill NE patients revealed three PUU strains related to but distinct from previously reported strains from Finland. The M-segment sequence of PUU from bank voles trapped near the probable site of infection for one of the patients showed 98.2% identity to that of the PUU strain obtained from the patient, suggesting a link between wild-type PUU from the natural focus and the NE case. The S-segment sequences from the patient and the bank voles, however, showed substantially lower identity (95.8%). As this difference in diversity for M and S genes (1.8 and 4.2%) is atypical for PUU genetic drift, one possibility is that the strain acquired at the putative place of infection is a reassortant one.
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| 9. |
- Sjolander, KB, et al.
(författare)
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Evaluation of serological methods for diagnosis of Puumala hantavirus infection (nephropathia epidemica)
- 1997
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Ingår i: Journal of clinical microbiology. - : American Society for Microbiology. - 0095-1137 .- 1098-660X. ; 35:12, s. 3264-3268
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Tidskriftsartikel (refereegranskat)abstract
- Nephropathia epidemica (NE), Puumala (PUU) virus infection, is a febrile disease which is commonly associated with acute renal impairment. To differentiate NE from other acute febrile illnesses, a rapid and reliable serological diagnosis is important, and a number of different protocols have recently been introduced. In the present report we describe a comparative evaluation of six PUU virus immunoglobulin M (IgM) and seven IgG enzyme-linked immunosorbent assay (ELISA) protocols based on native, Escherichia coli-expressed, or baculovirus-expressed nucleocapsid protein (N). Neutralization and immunofluorescence assays were included for comparison. Equally high sensitivities and specificities were obtained with three mu-capture-based IgM ELISAs using native, baculovirus-expressed, and E. coli-expressed N antigens, respectively, and by an ELISA based on purified E. coli-expressed full-length N adsorbed to solid phase. The assays based on truncated amino-terminal N proteins, including a commercially available PUU virus IgM ELISA, all showed lower sensitivities. For detection of PUU virus-specific IgG, ELISAs based on monoclonal antibody-captured native or baculovirus-expressed N antigens showed optimal sensitivities and specificities, while the assays based on E. coli-expressed N did not detect all PUU virus IgG-positive serum samples. A commercially available PUU virus IgG ELISA based on E. coli-expressed amino-terminal N showed a significantly lower sensitivity than those of all other IgG assays.
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| 10. |
- Vapalahti, O, et al.
(författare)
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Antigenic properties and diagnostic potential of puumala virus nucleocapsid protein expressed in insect cells
- 1996
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Ingår i: Journal of clinical microbiology. - : American Society for Microbiology. - 0095-1137 .- 1098-660X. ; 34:1, s. 119-125
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Tidskriftsartikel (refereegranskat)abstract
- Puumala virus (PUU) is a member of the genus Hantavirus in the family Bunyaviridae and the causative agent of nephropathia epidemica, a European form of hemorrhagic fever with renal syndrome. Sera of nephropathia epidemica patients react specifically with PUU nucleocapsid (N) protein. In order to safely provide large quantities of antigen for diagnostic purposes, PUU Sotkamo strain N protein was expressed by using the baculovirus system in Sf9 insect cells to up to 30 to 50% of the total cellular protein. The recombinant N protein (bac-PUU-N) was solubilized with 6 M urea, dialyzed, and purified by anion-exchange liquid chromatography. In an immunoglobulin M mu-capture assay purified and unpurified bac-PUU-N antigen showed identical results compared with the results of a similar assay based on native PUU antigen grown in Vero E6 cells. An immunoglobulin G monoclonal antibody-capture assay based on unpurified bac-PUU-N also showed results identical to those of an assay with native PUU-N antigen. Moreover, a panel of monoclonal antibodies reactive with eight different epitopes showed identical reactivity patterns with both natural and bac-PUU-N antigen, while two epitopes in PUU-N expressed as a fusion protein in Escherichia coli were not recognized. Puumala hantavirus N protein expressed by the baculovirus system offers a safe and inexpensive source of specific antigen for large-scale diagnostic and seroepidemiological purposes.
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