DNA vaccination with hantavirus M segment elicits neutralizing antibodies and protects against seoul virus infection.
Seoul virus (SEOV) is one of four known hantaviruses causing hemorrhagic fever with renal syndrome (HFRS). Candidate naked DNA vaccines for HFRS were constructed by subcloning cDNA representing the medium (M; encoding the G1 and G2 glycoproteins) or small (S; encoding the nucleocapsid protein) genome segment of SEOV into the DNA expression vector pWRG7077. We vaccinated BALB/c mice with three doses of the M or S DNA vaccine at 4-week intervals by either gene gun inoculation of the epidermis or needle inoculation into the gastrocnemius muscle. Both routes of vaccination resulted in antibody responses as measured by ELISA; however, gene gun inoculation elicited a higher frequency of seroconversion and higher levels of antibodies in individual mice. We vaccinated Syrian hamsters with the M or S construct using the gene gun and found hantavirus-specific antibodies in five of five and four of five hamsters, respectively. Animals vaccinated with the M construct developed a neutralizing antibody response that was greatly enhanced in the presence of guinea pig complement. Immunized hamsters were challenged with SEOV and, after 28 days, were monitored for evidence of infection. Hamsters vaccinated with M were protected from infection, but hamsters vaccinated with S were not protected. (+info
Cellular entry of hantaviruses which cause hemorrhagic fever with renal syndrome is mediated by beta3 integrins.
Hantaviruses replicate primarily in the vascular endothelium and cause two human diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). In this report, we demonstrate that the cellular entry of HFRS-associated hantaviruses is facilitated by specific integrins expressed on platelets, endothelial cells, and macrophages. Infection of human umbilical vein endothelial cells and Vero E6 cells by the HFRS-causing hantaviruses Hantaan (HTN), Seoul (SEO), and Puumala (PUU) is inhibited by antibodies to alphavbeta3 integrins and by the integrin ligand vitronectin. The cellular entry of HTN, SEO, and PUU viruses, but not the nonpathogenic Prospect Hill (PH) hantavirus (i.e., a virus with no associated human disease), was also mediated by introducting recombinant alphaIIbbeta3 or alphavbeta3 integrins into beta3-integrin-deficient CHO cells. In addition, PH infectivity was not inhibited by alphavbeta3-specific sera or vitronectin but was blocked by alpha5beta1-specific sera and the integrin ligand fibronectin. RGD tripeptides, which are required for many integrin-ligand interactions, are absent from all hantavirus G1 and G2 surface glycoproteins, and GRGDSP peptides did not inhibit hantavirus infectivity. Further, a mouse-human hybrid beta3 integrin-specific Fab fragment, c7E3 (ReoPro), also inhibited the infectivity of HTN, SEO, and PUU as well as HPS-associated hantaviruses, Sin Nombre (SN) and New York-1 (NY-1). These findings indicate that pathogenic HPS- and HFRS-causing hantaviruses enter cells via beta3 integrins, which are present on the surfaces of platelets, endothelial cells, and macrophages. Since beta3 integrins regulate vascular permeability and platelet function, these findings also correlate beta3 integrin usage with common elements of hantavirus pathogenesis. (+info
Polymerase chain reaction detection of Puumala virus RNA in formaldehyde-fixed biopsy material.
BACKGROUND: Infections with hantaviruses, mainly Clethrionomys-derived Puumala viruses, are known causes of acute renal failure [hemorrhagic fever with renal syndrome (HFRS)] in western Europe. Laboratory diagnosis is primarily based on serology. At the time of clinical symptoms, viral RNA can hardly be detected in the blood or urine, indicating that polymerase chain reaction (PCR) is of little diagnostic value for these infections. Biopsy material is usually formaldehyde-fixed and, thus, regarded as poor quality for PCR applications. The aim of this study was to establish a technique to retrieve such material for laboratory diagnostic. METHODS: Formaldehyde-fixed, paraffin-embedded kidney biopsies of 14 patients with renal failure either clinically suspected for HFRS (7 cases) or caused by unknown (2 cases) or known other causes (drugs, sarcoidosis; 5 cases) were histologically investigated. An established S segment-specific PCR assay was applied to RNA isolated from the biopsies, and amplification products were verified by direct sequence determination. RESULTS: Investigations revealed a typical histopathological appearance for hantavirus infections in all seven suspected HFRS cases and one case of unknown cause. With five of the suspected HFRS cases, hantavirus-specific RNA was detected. Sequence comparison revealed a close relationship to corresponding nucleoproteins of known Puumala viruses. CONCLUSION: The established technique provides a simple and powerful tool that expands the diagnostic possibilities, especially for otherwise unidentified or retrospective cases. It further allows insight into the molecular epidemiology of HFRS-causing agents. (+info
Human memory cytotoxic T-lymphocyte (CTL) responses to Hantaan virus infection: identification of virus-specific and cross-reactive CD8(+) CTL epitopes on nucleocapsid protein.
Hantaan virus, the prototypic member of the Hantavirus genus, causes hemorrhagic fever with renal syndrome in humans. We examined the human memory T-lymphocyte responses of three donors who had previous laboratory-acquired infections with Hantaan virus. We demonstrated virus-specific responses in bulk cultures of peripheral blood mononuclear cells (PBMC) from all donors. Bulk T-cell responses were directed against either Hantaan virus nucleocapsid (N) or G1 protein, and these responses varied between donors. We established both CD4(+) and CD8(+) N-specific cell lines from two donors and CD4(+) G1-specific cell lines from a third donor. All CD8(+) cytotoxic T-lymphocyte (CTL) lines recognized one of two epitopes on the nucleocapsid protein: one epitope spanning amino acids 12 to 20 and the other spanning amino acids 421 to 429. The CTL lines specific for amino acids 12 to 20 were restricted by HLA B51, and those specific for amino acids 421 to 429 were restricted by HLA A1. The N-specific CTL lines isolated from these two donors included both Hantaan virus-specific CTLs and hantavirus cross-reactive CTLs. Responses to both epitopes are detectable in short-term bulk cultures of PBMC from one donor, and precursor frequency analysis confirms that CTLs specific for these epitopes are present at relatively high precursor frequencies in the peripheral T-cell pool. These data suggest that infection with Hantaan virus results in the generation of CTL to limited epitopes on the nucleocapsid protein and that infection also results in the generation of cross-reactive T-cell responses to distantly related hantaviruses which cause the distinct hantavirus pulmonary syndrome. This is the first demonstration of human T-lymphocyte responses to Hantaan virus. (+info
Short report: simultaneous occurrence of Dobrava, Puumala, and Tula Hantaviruses in Slovakia.
The prevalence of antibody to hantaviruses in Slovakia (serum panel n = 2,133) was lower in the western part (0.54%) and higher in the eastern part (1.91%) of the country and was found to be significantly enhanced in a group of forest workers from eastern Slovakia (5.88%). One-third of the IgM-negative convalescent phase sera from patients with hemorrhagic fever with renal syndrome exhibited antibodies reacting predominantly with Puumala virus antigen, while two-thirds had antibodies directed mainly against Hantaan virus antigen. Fine analysis of two Hantaan virus-reactive sera by a focus reduction neutralization test showed that Dobrava hantavirus was the source of these human infections. Initial results of rodent screening indicated the circulation of Dobrava virus in populations of striped field mice (Apodemus agrarius) in eastern Slovakia. (+info
Hemorrhagic fever with renal syndrome in the Dolenjska region of Slovenia--a 10-year survey.
This report describes the first investigation of clinical findings for a larger series of patients with hemorrhagic fever with renal syndrome (HFRS) who were infected with Dobrava virus. From 1985 to 1995, 38 patients with serologically confirmed HFRS were hospitalized at the regional hospital in Novo mesto in the Dolenjska region of Slovenia. On the basis of results of serological examination, 24 patients had Dobrava virus infection, and 14 patients had Puumala virus infection. Complete clinical data were available for 31 patients. Eleven patients underwent hemodialysis for treatment of acute oliguric or anuric renal failure. Four patients, all infected by Dobrava virus, had signs of shock and severe bleeding. Three severely ill Dobrava virus-infected patients died of hemorrhagic complications. We have demonstrated that Dobrava and Puumala viruses coexist in a single region of endemicity and are capable of causing HFRS with significant differences in severity. (+info
Diagnostic potential of puumala virus nucleocapsid protein expressed in Drosophila melanogaster cells.
Puumala virus (PUU) nucleocapsid protein (N) was expressed in insect cells by using the Drosophila Expression System (DES; Invitrogen BV, Groningen, The Netherlands). Stable transfectants were established by hygromycin B selection and showed continuous expression of the recombinant protein (DES-PUU-N) for at least 5 months. The antigenic property of DES-PUU-N was shown to be identical to that of native PUU N when examined with a panel of hantavirus-specific monoclonal antibodies. Enzyme-linked immunosorbent assays (ELISAs) for detection of human immunoglobulin M (IgM) and IgG antibodies were established by using DES-PUU-N as antigen and were compared to assays based on native N. The ELISAs were evaluated for patient diagnosis and seroepidemiological purposes with panels of sera collected from patients with hemorrhagic fever with renal syndrome (HFRS) and from healthy blood donors. Equally high sensitivities and specificities for detection of PUU-specific IgM in acute-phase HFRS patient sera were obtained by the ELISA based on DES-PUU-N and the assay based on the native antigen. For detection of PUU-specific IgG, the ELISA based on monoclonal antibody-captured DES-PUU-N antigen showed optimal sensitivity and specificity. (+info
Systemic inflammation in hemorrhagic fever with renal syndrome correlates with hypotension and thrombocytopenia but not with renal injury.
Systemic inflammation is common in patients with nephropathia epidemica (NE), a European form of hemorrhagic fever. Markers of inflammation were studied in a patient with NE with respiratory insufficiency (patient 1), 18 other patients with NE, and 13 patients with a viral infectious disease other than NE. Neutrophil and monocyte CD11b expression levels, determined by flow cytometry; soluble interleukin (IL)-2 receptor (sIL-2R), IL-6, and IL-8 concentrations, determined by means of Immulite; and soluble E-selectin, determined by ELISA, were higher in patients with NE than in healthy subjects. The findings were not specific for NE and did not correlate with serum creatinine levels, but the findings correlated inversely with mean arterial pressure (sIL-2R and monocyte CD11b expression) and minimum platelet count (sIL-2R, IL-6, neutrophil, and monocyte CD11b expression). Monocyte CD11b expression in patient 1 was extremely high, suggesting that monocytes may contribute to development of lung injury. Severity of inflammation in patients with NE is related to hypotension and platelet consumption but not to renal injury. (+info