Diss. (sammanfattning) Umeå : Umeå universitet, 2005. / Härtill 4 uppsatser.

No description available.

Puumala hantavirus (PUUV) causes hemorrhagic fever with renal syndrome in Europe. After inhalation of virus shed by bank voles, the virus systemically targets the vascular endothelium leading to vascular dysfunction and leakage. Many patients with PUUV infection experience cardiopulmonary manifestations but the underlying mechanisms have not been determined. The aims of the studies presented were to describe cardiopulmonary manifestations, investigate pathogenetic mechanisms including presence of virus in the lungs and the local immune response in PUUV infection. The results showed cardiopulmonary involvement of varying severity in almost all studied patients. High-resolution computed tomography frequently revealed vascular leakage into the lungs or pleural cavities. Pulmonary function tests generally showed reduced gas diffusing capacity, evidenced in patients as dyspnea, poor oxygenation and frequent need of oxygen treatment. Among patients who were not fully recovered at 3 months follow-up, remaining decreased gas diffusing capacity was highly common. Echocardiography revealed mainly right heart dysfunction which was related to manifestations within the lungs, in terms of increased estimated pulmonary vascular resistance, mild to moderate pulmonary hypertension, and reduced right ventricular systolic function in patients with more pronounced lung involvement, as indicated by need of oxygen treatment. Analyses on bronchoalveolar lavage (BAL) and bronchial biopsies revealed a highly activated cytotoxic T cell (CTL) response in the lungs. The CTL response was not balanced by the expansion of regulatory T cells and high numbers of CTLs were associated with more severe disease. PUUV RNA was detected in almost all patients’ BAL samples and the viral load was inversely correlated to the number of CTLs. Three patients presenting with severe and fatal cardiopulmonary distress were also described. Autopsies revealed PUUV protein in vascular endothelium in all investigated organs, including the heart and lungs, along with a massive CTL response mainly in the lungs. In conclusion, cardiopulmonary involvement of varying severity was present in almost all patients with PUUV infection. Cytotoxic immune responses could contribute to disease development but also help in clearing the infection. Long lasting fatigue after hantavirus infection may be explained by remaining manifestations within the lungs.

Hantaviren (Familie Bunyaviridae) sind Erreger, die von Nagetieren auf den Menschen übertragen werden und Hämorrhagische Fieber mit Renalem Syndrom (HFRS) auslösen. Die vorgelegte Arbeit beinhaltet derartige Ergebnisse zu zwei europäischen Hantaviren, dem Dobravavirus (DOBV) und dem Tulavirus (TULV). DOBV ist ein wichtiger HFRS-Erreger in Europa. DOBV Stämme kommen in mindestens zwei Nagerspecies, der Gelbhalsmaus (Apodemus flavicollis) und der Brandmaus (A. agrarius) vor. In Übereinstimmung mit diesen natürlichen Wirten bilden die Virusstämme zwei genetische Linien: DOBV-Af und DOBV-Aa. Die phylogenetischen Analysen von den Nukleotidsequenzen der S-, M- und L-Segmente von sympatrisch vorkommenden DOBV-Af und DOBV-Aa Stämmen aus Mitteleuropa zeigten das Vorkommen von Reassortmentprozessen der Genomsegmente während der Evolution der Virusspecies. Ausserdem, wurde die virale Nukleotidsequenz aus einem DOBV-seropositiven HFRS-Patienten aus Detschland amplifiziert. Damit wurde erstmalig der molekulare Beweis erbracht, dass DOBV in Mitteleuropa HFRS auslöst und dass die DOBV-Aa Linie humanpathogen ist. Aus einer in der Slowakei gefangenen A. agrarius Maus haben wir ein neues Virusisolat gewonnen, welches "Slovakia (SK/Aa)" genannt wurde. SK/Aa ist das bisher einzige Virusisolat, das die DOBV-Aa Linie repräsentiert. Es wurde gemeinsam mit einem Isolat der DOBV-Af Linie zur vergleichenden Typisierung der Antikörper von mitteleuropäischen HFRS-Patienten mittels Fokusreduktionsneutralisationstest eingesetzt. Die Seren der meisten Patienten zeigten die höchsten neutralisierenden Antikörpertiter gegenüber SK/Aa, was die Schlussfolgerung zulässt, dass DOBV-Aa Stämme für die meisten DOBV-Infektionen in Mitteleuropa verantwortlich sind. TULV wird durch die Feldmaus (Microtus arvalis) beherbergt. Die Fähigkeit zur Auslösung von HFRS war bisher wenig bekannt. Wir haben den ersten Fall von HFRS gefunden, der mit einer TULV Infektion assoziiert ist. Aus demselben geographischen Gebiet in Nordostdeutschland konnten aus Feldmäusen TULV Nukleotidsequenzen amplifiziert werden. In phylogenetischen Analysen clustern sie mit Stämmen aus Polen und bilden mit diesen gemeinsam eine eigene, neue genetische Linie. Ausser dem hier untersuchten DOBV und dem länger bekannten Puumalavirus ist TULV offenbar das dritte Hantavirus, das in Mitteleuropa HFRS hervorruft. / Hantaviruses (Bunyaviridae family) are rodent-borne bunyaviruses that cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia. This thesis presents novel data about two European hantaviruses, Dobrava virus (DOBV) and Tula virus (TULV). DOBV is an important etiologic agent of HFRS in Europe. DOBV strains were found to be hosted by at least two different rodent species, yellow-necked mouse (Apodemus flavicollis) and striped field mouse (A. agrarius). According to their natural hosts they form the distinct genetic lineages DOBV-Af and DOBV-Aa, respectively. We have determined and analysed the complete S and M, and partial L segment nucleotide sequences of sympatrically occurring DOBV-Af and DOBV-Aa strains from Central Europe. Molecular phylogenetic analyses gave evidence for genetic reassortment in the evolution of the virus species. Moreover, we amplified a DOBV-Aa nucleotide sequence from a DOBV-seropositive HFRS patient from Germany. This is the first molecular identification of human infection by DOBV in Central Europe and the first direct proof that a virus strain related to the DOBV-Aa lineage, carried by A. agrarius rodents, is able to cause HFRS. Under biosafety level 3 conditions, we have established a DOBV isolate named Slovakia (SK/Aa) from an A. agrarius animal captured in Slovakia. SK/Aa, as the only isolate clearly belonging to the DOBV-Aa lineage, can be taken as the representative of this virus lineage. The new virus isolate, in comparison to a DOBV-Af strain, was used for serotyping neutralising antibodies of HFRS patients in Central Europe by the use of a focus reduction neutralisation assay. Most patients'' sera exhibited a higher end-point titer towards SK/Aa suggesting that DOBV-Aa strains are responsible for most of the DOBV HFRS cases in this region. TULV is carried by European common voles (Microtus sp.). Its pathogenic potential for humans was rather unknown. We have described the first case of HFRS which can be associated with TULV infection. Moreover, TULV strains detected in M. arvalis near the home village of the patient in North-East Germany clustered with strains from Poland and represent a new, well-supported genetic lineage within the TULV species. In addition to DOBV and longer known Puumala virus, TULV is most likely an additional causative agent of HFRS in Central Europe.

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.

Ο ιός του αιμορραγικού πυρετού Κριμαίας-Κογκό (Crimean-Congo Hemorrhagic Fever Virus, CCHFV), καθώς και οι χανταϊοί (hantaviruses) προκαλούν στον άνθρωπο αιμορραγικό πυρετό. Αυτοί παρουσιάζουν ευρεία γεωγραφική κατανομή και αποτελούν απειλή για τη δημόσια υγεία, λόγω του υψηλού ποσοστού θνητότητας που σημειώνουν και της απουσίας αποτελεσματικής θεραπευτικής αγωγής. Παρότι επιδημιολογικές μελέτες δείχνουν την παρουσία αντισωμάτων στον ελληνικό πληθυσμό, περιορισμένες είναι οι αναφορές κλινικών περιστατικών CCHF και HFRS στην Ελλάδα. Σκοπός της παρούσας μελέτης είναι να προσδιορίσει τον επιπολασμό της μόλυνσης με τον CCHFV και τους χανταϊούς στον Ν. Αχαΐας, που αν και παρουσιάζει ευνοϊκές συνθήκες για την κυκλοφορία των δύο ιών, δεν έχει μελετηθεί στο παρελθόν. Σχεδιάσθηκε διατμηματική μελέτη και συγκεντρώθηκαν προοπτικά 207 δείγματα ορού φαινομενικά υγιών ατόμων-κατοίκων της περιοχής, τα οποία εξετάστηκαν με τη μέθοδο ELISA και έμμεσου ανοσοφθορισμού για την ύπαρξη αντισωμάτων έναντι του CCHFV και των χανταϊών. Ο επιπολασμός για τη μόλυνση με CCHFV βρέθηκε 3,4% και 9,7% για τη μόλυνση με χανταϊούς, ενώ κανένα από τα οροθετικά άτομα δεν ανακαλούσε συμπτώματα παρόμοια με αυτά του CCHF ή του HFRS. Για τον CCHFV, βρέθηκε ότι η ηλικία, η αγροτοκτηνοτροφική ενασχόληση, η κατοχή/εκτροφή αιγοπροβάτων, το ιστορικό νύγματος κρότωνα, η μόνιμη διαμονή σε υψόμετρο ≥400μ., η μόνιμη διαμονή σε μη αρδευόμενες αρόσιμες εκτάσεις ή σε αγροτικές εκτάσεις με σημαντικό ποσοστό φυσικής βλάστησης, καθώς και η μόνιμη διαμονή σε αγροτική περιοχή είναι σημαντικοί παράγοντες κινδύνου. Από αυτούς, το νύγμα κρότωνα, η αγροτοκτηνοτροφική ενασχόληση και η μόνιμη διαμονή σε υψόμετρο ≥400μ. βρέθηκαν να προβλέπουν καλύτερα την οροθετικότητα ενός ατόμου. Επίσης, βρέθηκε ότι παράγοντες που σχετίζονται με τη μόλυνση με χανταϊούς είναι: η ηλικία, η θέαση τρωκτικών σε ακτίνα <200μ. γύρω από την οικία και η ιδιοκτησία υπόγειας αποθήκης. Από αυτούς, μόνο η ηλικία βρέθηκε να προβλέπει καλύτερα την οροθετικότητα ενός ατόμου. Επιπλέον, παρατηρήθηκε ότι σχεδόν το 75% των θετικών ατόμων για αντισώματα έναντι των χανταϊών παρουσίαζαν ήπια επηρεασμένη νεφρική λειτουργία. Εντοπίστηκαν, επίσης, ενδημικές εστίες των ιών στον νομό: ο Δ. Ερυμάνθου για τον CCHFV και ο Δ. Δυτικής Αχαΐας για του χανταϊούς. Λαμβάνοντας υπόψη τα παραπάνω αποτελέσματα, θα πρέπει οι κλινικοί γιατροί της περιοχής να συμπεριλαμβάνουν τον CCHF και τον HFRS στη διαφορική διάγνωση εμπύρετων νοσημάτων, ιδίως όταν αυτά συνοδεύονται από θρομβοπενία ή επηρεασμένη νεφρική λειτουργία. / Crimean-Congo hemorrhagic fever virus (CCHFV) and hantaviruses cause to humans fever with hemorrhagic manifestations. These viruses present wide geographic distribution and represent major threats for public health, because of the high fatality rate that they present and the lack of appropriate treatment. Although seroprevalence studies show the presence of antibodies against CCHFV and hantaviruses in the greek population, only some reports of human cases have been reported to date in Greece. The aim of the present study is to estimate seroprevalence for CCHFV and hantaviruses in humans in the prefecture of Achaia, where the local conditions potentially favor the circulation of these viruses and which has not been previously studied. A cross-sectional study was designed and 207 human sera were collected from apparently healthy individuals living in Achaia, which were tested for CCHFV and hantaviruses IgG antibodies by ELISA and by indirect immunofluorescence assay (IFA). Seroprevalence for CCHFV infection was estimated at 3.4%, whereas for hantaviruses at 9.7%; none recalled any illness resembling CCHF or HFRS. For CCHFV, it was found that age, agro-pastoral occupation, tending sheep and/or goats, tick bite, living in areas at an altitude of ≥400m., living at rural areas, living on non-irrigated arable land or on land principally occupied by agriculture, with significant areas of natural vegetation are significantly related to seropositivity. Among them, tick bite, agro-pastoral occupation and living in areas at an altitude of ≥400m. better predict seropositivity of an individual. For hantaviruses, it was found that age, rodent sighting around home and the ownership of an underground shed are significantly related to seropositivity. Among them, it seems that only age can predict seropositivity of an individual. Moreover, it was observed that almost 75% of the seropositive for hantaviruses individuals presented mild renal dysfunction. In this study, endemic foci were also detected: the municipality of Erimanthos for CCHFV and the municipality of Western Achaia for hantaviruses. Clinicians should include CCHF and HFRS in the differential diagnosis of an acute febrile case, especially when thrombocytopenia or impaired renal function is encountered.