Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 5 uppsatser.

Rodent-borne hantaviruses (family Bunyaviridae) cause two distinct human infections; hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). HFRS is a common viral zoonosis, characterized by fever, renal dysfunction and hemostatic imbalance. Four HFRS-associated hantaviruses have been described: Hantaan virus and Seoul virus mainly found in Asia, Dobrava virus, encountered in the Balkan region and Puumala virus (PUU), causing mild HFRS (nephropathia epidemica; NE) in Europe. HPS, recently discovered in the Americas, involves adult respiratory distress syndrome with a high mortality rate and is caused by Sin Nombre virus. Hantaviruses are enveloped and carry a RNA genome which encodes a polymerase, two glycoproteins and a nucleocapsid protein. The latter elicits a strong humoral immune response in infected patients. The clinical diagnosis of hantavirus infections has until recently relied on serological confirmation by immunofluorescense assay (IFA) and enzyme-linked immunosorbent assay (ELISA) using cell culture derived viral antigens. Due to the hazardous nature of hantaviruses and variable virus yield in cell culture we aimed at using recombinant hantavirus proteins for serological purposes. We expressed PUU N in E. coli (PUU rN) and found that high levels of IgM to this protein could be detected at onset of NE. This indicated that it was useful as the sole antigen for serodiagnosis. Our finding was confirmed by comparing IFA and PUU rN ELISA using 618 sera collected at the regional diagnostic laboratory. Full-length PUU rN is difficult to purify due to aggregation to E. coli remnants. We therefore located the important domain for the humoral immune response by utilizing truncated PUU rN proteins to its amino-terminal region (amino acid 7-94). Amino acid 1-117 of N of the five major human hantavirus pathogens were produced in E. coli. Serological assays based on them could detect IgM and IgG serum responses in 380 HFRS and HPS patients from Sweden, Finland, Slovenia, China, Korea and the USA with high sensitivity. In an epidemiological investigation of hantavirus serum responses in European Russia we unexpectedly found antibody responses to the hantaviruses found in east Asia and the Balkan region in 1.5 %, speaking in favour for the presence of such virus in this region. The degree of cross reactivity within the hantavirus genus was adressed by following the serum responses in NE patients. We found an increase of cross reactivity during the maturation of the immune response from onset of disease up to three years by comparing the IgG reactivity towards the hantavirus aminoterminal rN proteins. The first human isolate of the causative agent of NE in Scandinavia was recovered in cell culture from phytohemagglutinin stimulated leukocytes. Serological analysis revealed that this virus belongs to the PUU hantavirus serotype, distinct from the rodent prototype PUU Sotkamo. The human PUU Umeå is unique but genetically similar to rodent isolates from northern Sweden. / digitalisering@umu.se

A hantavirose (infecção por Hantavírus) é uma das zoonoses que vem preocupando as autoridades sanitárias de todo o mundo. Sua ocorrência se deve principalmente os distúrbios ecológicos é transmitida ao homem através de inalação de partículas virais contida na excreta de roedores. São conhecidas duas doenças humanas distintas causadas pelo Hantavírus: a Febre Hemorrágica com Síndrome Renal (FHSR) e a Síndrome Pulmonar e Cardiovascular (SPCVH). O objetivo deste estudo foi verificar a ocorrência de anticorpos IgG anti-hantavírus, através do ELISA, em populações da Amazônia e Sudeste brasileiro, que vivem em contato com os roedores silvestres, utilizando a proteína recombinante do vírus Araraquara expressa em Escherichia coli. Do total de estudados 1308 soros humanos estudados, na Amazônia (1078) encontramos 59 soros positivos (5%). Na cidade Machadinho do OesteRO os soros coletados durante o ano 2003, foram analisados 638, onde foram encontrados 20 soros positivos (4,5%); e no Rio Machado RO. foram analisados 435 soros da população ribeirinha onde foram encontrados 39 (5%) soros positivos, respectivamente. Após análise realizada em 151 soros humanos provenientes do Vale do Ribeira, em 2007; e 84 no Pontal do Paranapanema, em 2008, foram observados 14 positivos (9%) e 6 (7%) das amostras, respectivamente. / The genus Hantavirus of the family Bunyaviridae includes a large number of rodent-borne viruses that are distributed worldwide. The occurrence is due mainly to ecological disturbances and it is transmitted to the humans through inhalation of virus particles contained in the excreta of wild rodents. Two different human diseases known to be caused by Hantavirus: are Hemorrhagic Fever with Renal Syndrome (HFRS) and Hantavirus Cardiopulmonary Syndrome (HPS). The main objective of this study was detected antibody against hanatavirus (IgG) by ELISA, in Amazon region and Brazilian Southwest populations who live in contact with the wild rodents, using recombinant protein (antigen) of the Araraquara virus expressed in Escherichia coli. We study 1308 human sera (1078 from Amazon region) and there were found 59 (5%) positive sera. From the city of Machadinho do Oeste RO (2003 year), 633 sera were analysed, where there were found to be 20 positive (4.5%) serums. In Machado river RO (2005 year), 435 sera of the river-dwelling population were analysed where there were found 39 (5%) positive sera, respectively. After analysis was accomplished for 151 human sera coming from the Vale do Ribeira - SP, in 2007, and 84 from the Pontal do Paranapanema - SP, in 2008, 14 (9%) and 6 (7%) of the samples were observed to be positive, respectively.

Hantaviruses are members of the virus family Bunyaviridaethat cause two potentially life-threatening diseases in humans: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (BPS). HFRS is caused by Old World hantaviruses that are endemic in many Asian and European countries. Infections with Old World hantaviruses can range in severity from asymptomatic to moderate or severe, depending primarily on the infecting serotype of virus. HPS is caused by New World hantaviruses in North and South America. New World hantaviruses are rarely asymptomatic and are severe in the majority of cases. These syndromes are distinct from one another in the primary target organ of virus infection (kidney vs. lung), but have important clinical features in common, including fever, thrombocytopenia, and a capillary leak syndrome. These common clinical manifestations suggest that the underlying mechanisms of disease may be similar in the two syndromes. The precise mechanisms of pathogenesis of HFRS and HPS are poorly characterized, but may be mediated in part by immunopathology. Hantaviruses are able to establish infections in many human cell types, including primary human endothelial cells, without having any cytopathic effect on these cells. Human infections with hantavirus result in a robust activation of the humoral and cellular immune response, and we hypothesize that these immune responses contribute to the pathology of disease. Evidence for the activation of T lymphocytes, and their potential involvement in immunopathology, includes increases in the number of circulating, activated CD8+ T cells during HFRS, the presence of lymphocytic infiltrates (predominantly CD8+T cells) in kidney biopsies from patients with acute HFRS, and associations between certain HLA haplotype and disease severity following hantavirus infection. This thesis is the first examination of human T lymphocyte responses that are generated during HFRS. Initially, we studied memory T cell responses in scientists who were sub-clinically infected with Hantaan virus (HTNV), the prototype hantavirus. We later investigated memory T cell responses in healthy Finnish adults who had HFRS caused by Puumala virus (PUUV), a hantavirus endemic primarily in Scandinavia. At the onset of these studies, there was no available information on human T lymphocyte responses to Old World hantaviruses. Virus-specific CD8+ and CD4+human T cell lines had been isolated from patients with acute HPS caused by Sin Nombre virus (SNV) infection. In that study, conducted in our laboratory, several human T cell epitopes on the nucleocapsid (N) protein and G2 envelope glycoprotein of SNV were identified and characterized. We decided to perform similar analyses on PBMC from donors who had been infected with HTNV and PUUV, in order to determine the specificity and diversity of the T cell response to Old World hantaviruses. The initial study of three donors who had sub-clinical infections with HTNV demonstrated that virus-specific T cell responses could be detected in all the donors following in vitro stimulation of PBMC with inactivated virus. In two of the donors, the virus-specific cytolytic T cells (CTL) recognized the HTNV N protein, and in the third donor the virus-specific CTLs recognized the HTNV G1 glycoprotein. Isolation and characterization of virus-specific T cells from two donors resulted in the identification of two CD8+ T cell epitopes on the HTNV N protein, which were restricted by either HLA A1 or B51. These CTL lines included both HTNV-specific (HLA B51-restricted) and serotype-cross reactive (HLA A1 restricted) lines. In one subject, these virus-specific T cell responses were detectable in IFN-γ ELISPOT assays following peptide stimulation, and in bulk cultures after short-term stimulation with inactivated HTNV. These results indicated that the CD8+CTL responses of humans after sub-clinical infection with HTNV were readily detectable and were directed against a limited number of viral proteins and epitopes. In addition, sub-clinical infection resulted in the generation of both virus-specific and cross-reactive CTL responses. We reasoned that hantavirus infections that lead to clinical illness may result in the generation of more robust and/or diverse virus-specific T cell responses than in sub-clinical infections. To address this question, we studied the memory CD8+ T cell responses in a group of healthy adults from Finland who had HFRS caused by PUUV infection between the years 1984 and 1995. We detected virus-specific CTL in the bulk cultures of seven of eleven immune individuals tested following stimulation with infectious virus. The PUUV proteins N, G1 and G2 were recognized by CTLs in six, five, and two donors respectively. Extensive cloning of T cells from two donors resulted in the isolation of sixty-three virus-specific CTL lines, the majority of which (61/63) were specific for the PUUV N protein. Six novel CD8+ CTL epitopes and one CD4+ CTL epitope were identified on the N protein, all of which clustered in the center of the protein between amino acids 173 and 251. The CTL lines specific for these epitopes were restricted by a variety of HLA alleles including A2, A28, B7 and B8, and were primarily serotype specific when tested against target cells expressing HTNV or SNV N protein. IFN-γ ELISPOT analysis using the defined epitopes to stimulated PBMC, revealed high frequencies of circulating N-specific CD8+ T cells in eight of thirteen individuals tested. Finally, T cell receptor (TCR) Vβ analysis of CTL clones specific for one epitope (N204-12) demonstrated that cells in this population expressed up to five different Vβ chains. These results demonstrated that the PUUV N protein may be the dominant target of the CTL response, that the N-specific CD8+ CTL responses are diverse, heterogeneous, and primarily serotype specific, and that virus-specific memory CD8+T cells can persist at high levels for up to 15 years after the primary infection. In order to understand the pathology of HFRS and HPS, we must be able to assess the contribution of various factors that could potentially contribute to disease. The virus burden in the infected individual is likely to be an important factor in the severity of the resulting disease. Quantitative RT-PCR analysis of plasma samples from acute HPS patients demonstrated that a higher virus burden (as reflected by viral RNA copy number) is associated with more severe HPS. In order to perform similar analyses in patients with HFRS caused by PUUV, we established a quantitative RT-PCR assay for the detection of PUUV S segment RNA in patient plasma. The design and optimization of the PUUV-specific RT-PCR is described in this report. This assay will allow us to measure the virus burden in patients and compare these data with levels of T cell activation and with parameters of disease severity. In this way, we hope to gain an understanding of the kinetics and magnitude of both the virus burden and virus-specific T cell response during the acute illness. This thesis provides the first description of human virus-specific T cell responses to HTNV and PUUV. These data shed light on the nature of the CD8+ T cell responses that are generated following natural infections with PUUV and sub-clinical infections with HTNV. The studies of memory CD8+ T cell responses to PUUV, and the development of a PUUV-specific quantitative RT-PCR assay, establish the framework for future studies of the immunopathology of acute HFRS. Quantitative analysis of both virus burden and T cell responses during acute illness will provide insight into their relative contributions to the pathology of disease.

A hantavirose (infecção por Hantavírus) é uma das zoonoses que vem preocupando as autoridades sanitárias de todo o mundo. Sua ocorrência se deve principalmente os distúrbios ecológicos é transmitida ao homem através de inalação de partículas virais contida na excreta de roedores. São conhecidas duas doenças humanas distintas causadas pelo Hantavírus: a Febre Hemorrágica com Síndrome Renal (FHSR) e a Síndrome Pulmonar e Cardiovascular (SPCVH). O objetivo deste estudo foi verificar a ocorrência de anticorpos IgG anti-hantavírus, através do ELISA, em populações da Amazônia e Sudeste brasileiro, que vivem em contato com os roedores silvestres, utilizando a proteína recombinante do vírus Araraquara expressa em Escherichia coli. Do total de estudados 1308 soros humanos estudados, na Amazônia (1078) encontramos 59 soros positivos (5%). Na cidade Machadinho do OesteRO os soros coletados durante o ano 2003, foram analisados 638, onde foram encontrados 20 soros positivos (4,5%); e no Rio Machado RO. foram analisados 435 soros da população ribeirinha onde foram encontrados 39 (5%) soros positivos, respectivamente. Após análise realizada em 151 soros humanos provenientes do Vale do Ribeira, em 2007; e 84 no Pontal do Paranapanema, em 2008, foram observados 14 positivos (9%) e 6 (7%) das amostras, respectivamente. / The genus Hantavirus of the family Bunyaviridae includes a large number of rodent-borne viruses that are distributed worldwide. The occurrence is due mainly to ecological disturbances and it is transmitted to the humans through inhalation of virus particles contained in the excreta of wild rodents. Two different human diseases known to be caused by Hantavirus: are Hemorrhagic Fever with Renal Syndrome (HFRS) and Hantavirus Cardiopulmonary Syndrome (HPS). The main objective of this study was detected antibody against hanatavirus (IgG) by ELISA, in Amazon region and Brazilian Southwest populations who live in contact with the wild rodents, using recombinant protein (antigen) of the Araraquara virus expressed in Escherichia coli. We study 1308 human sera (1078 from Amazon region) and there were found 59 (5%) positive sera. From the city of Machadinho do Oeste RO (2003 year), 633 sera were analysed, where there were found to be 20 positive (4.5%) serums. In Machado river RO (2005 year), 435 sera of the river-dwelling population were analysed where there were found 39 (5%) positive sera, respectively. After analysis was accomplished for 151 human sera coming from the Vale do Ribeira - SP, in 2007, and 84 from the Pontal do Paranapanema - SP, in 2008, 14 (9%) and 6 (7%) of the samples were observed to be positive, respectively.