Cytotoxic T Lymphocytes (CTL) are known to play an important role in clearing infectious virus from infected hosts in a variety of viral infections. Depending on the type of virus and mode of virus entry both class I and class II restricted CTL can contribute to protection from virus-induced disease. Although CD8 positive CTL are associated with virus elimination and control in many viral infections, elimination of neurotropic viruses from the Central Nervous system (CNS) is more complex due to the lowered expression of MHC antigens on neuronal cells. This failure to constitutively express high levels of MHC antigens by neurons could serve as an advantage to avoid damage to this differentiated and non-renewable tissue. However, abnormal induction of MHC antigens in the CNS mediated by CD4 positive lymphocytes or by astrocytes have also been shown to cause destructive inflammation in the CNS. The present study deals with CTL responses against one such neurotropic virus called Japanese Encephalitis Virus (JEV). JEV is a positive-stranded RNA virus that belongs to the flavivirus group, a group that is among the most important agents causing human encephalitis worldwide. Although passive transfer of monoclonal antibodies against this virus has been shown to confer protection of mice from lethal challenge with virus, neither the presence of CTL against this virus nor its role in conferring protection has been reported so far. Understanding the CTL responses against these viruses acquired importance in light of recent reports that neurovirulence of JEV and yellow fever viruses can be enhanced by the administration of virus specific antibodies. Hence this study was undertaken to examine the possibility of raising CTL specific to JEV. The specificity of the CTL raised, their therapeutic value and the ability of different lymphocyte subsets to mediate protection in vivo are dealt with in this study. Generation of CTL against JEV The generation of CTL against JEV in BALB/c mice, requires MHC defined cell lines that not only support virus infection but are also histocompatible. Several cell lines were initially examined for their ability to support JEV infection as a prc-rcquisitc before their utilization in in vivo and in vitro stimulation protocols aimed at generating JEV-specific CTL. Virus infection was monitored by immunofluorescence using JEV envelope-specific monoclonal antibodies as well as by titration of virus produced from infected cells by plaque assays. These different cell lines that were characterised for their ability to support JEV infection were then utilised to generate and monitor antiviral CTL. Several in vivo immunisation protocols were examined initially find out which of these infected cells prime BALB/c mice efficiently for generation of virus-specific CTL upon secondary stimulation in vitro with infected syngeneic cells. Immunisation of mice with infected cells per se was preferred over free virus since this was thought to facilitate priming against some viral non-structural proteins preferentially found on infected cells in addition to other viral structural proteins. It was observed that not only infected syngeneic and allogeneic cells but also infected xenogeneic cells prime BALB/c mice for the generation of JEV- specific CTL upon secondary restimulation in vitro. An optimal protocol was standardised for the generation of CTL against JEV. This included primary in vivo immunisation of mice followed by secondary in vitro restimulation of splenocytes with infected syngeneic cells. Either immunisation alone or in vitro stimulation of naive splenocytes alone was unsuccessful. The effector cells generated specifically lysed JEV-infccted P388D1 targets but not uninfected P388D1 or YAC-1 targets suggesting that the lysis on infected targets is not mediated by Natural Killer activity. Specificity and MHC restriction of anti JEV Effectors Cell depletion studies using complement mediated lysis were performed to examine the phenotype of the cells mediating virus specific lysis of infected targets. Depletion of Lyt 2.2+ or Thy 1+ but not L3T4+ sub-populations of effector cells inhibited lysis of infected targets showing that the effectors mediating virus-specific lysis were Lyt-2+ T cells. Examination of target specificities and MHC restriction of the antiviral CTL generated showed that although infected xenogeneic cells were used for immunisation, the effector cells recognised only infected syngeneic (P388D1, Sp2/0) and semisyngeneic (Neuro 2a, YAC-1) cells. Virus-specific recognition was found to be class I Kd and class I Dd restricted. These effector cells were also found to recognise cells infected with a closely related flavivirus, West Nile Virus (WNV) suggesting that they were crossreactive to some degree. Based on the consensus motif that has been established for H-2Kd associated peptides, several nonamers were predicted as possible CTL epitopes by scanning the deduced amino acid sequences of three strains of JEV and WNV. Among several predicted nonamers, three peptides were examined for their ability to reconstitute lysis of uninfected targets by polyclonal anti JEV CTL populations. Results demonstrate that peptides derived from NS1 and NS3 but not NS5 protein of JEV were able to partially reconstitute lysis of uninfected targets by effectors when pulsed with the appropriate peptide. Protective ability of the CTL raised against JEV To examine whether anti-JEV effectors raised in vitro could confer protection from intracerebral challenge with JEV, these effectors were adoptively transferred into adult BALB/c mice intracerebrally along with 10 x LDJ0 dose of JEV. More than 55% of these animals were protected from death and survived beyond 100 days after JEV challenge demonstrating that adoptively transferred anti-JEV effectors could indeed confer protection from lethal challenge with JEV. However, adoptive transfer of effectors by either intravenous or intraperitoneal routes did not protect adult mice from the lethal effects of intracerebral challenge with JEV. In contrast to adult mice, newborn mice were not protected from death by the adoptively transferred effector cells. This was also supported by experiments where a correlation was observed with the increasing age of mice and the success of protection conferred by the adoptively transferred effector cells. To establish the identity of cell subsets responsible for protection, Lyt 2, L3T4 or Thy 1 positive cells were specifically depleted from the polyclonal CTL by multiple cycles of complement mediated lysis and the remaining cells were adoptively transferred intracerebrally along with 10 x LD of JEV. These results demonstrate that both Lyt 2 and L3T4 positive T cells present in the effector population were necessary to confer protection of adult mice. Examination of virus-specific neutralising antibodies in the sera of protected and unprotected mice revealed that presence of L3T4 positive cells in the adoptively transferred population increases virus-specific neutralising antibodies. However presence of neutralising antibodies alone was not sufficient to confer protection. The protection required both Lyt-2 and L3T4 positive cells together. These studies could in the long term throw some light on similar observations about age dependant susceptibility to JEV in humans.

In den letzten Jahren erreichten viele neue (emerging) Viren Europa, die zum Teil (z.T.) zoonotisch auf den Menschen übertragbar sind. So musste man sich mit Geflügel- und Schweinegrippe, Blauzungenkrankheit, Infektiöser Anämie der Einhufer oder auch SARS (severe acute respiratory syndrome) auseinandersetzen. Bedingt durch verschiedene Faktoren, wie Klimawandel oder zunehmende Globalisierung und damit einhergehendem Verkehr zwischen den Kontinenten verbesserten sich auch die Bedingungen für die Virusverbreitung, so dass viele für Deutschland untypische Krankheitserreger auch hier auftraten. Das West-Nil-Virus (WNV) ist in Europa bereits endemisch verbreitet und könnte somit eine besondere Gefahr für Deutschland darstellen. Es ist ein bekannter Zoonose-Erreger, und sein Eintrag und die rasche Verbreitung des Virus in Amerika 1999 zeigten wie gefährlich neue Viren in naiven Populationen sein können. Über die Verbreitung des Virus in Deutschland gibt es nur wenige Studien z.B. des Robert-Koch-Instituts (LINKE et al. 2007a) und des Friedrich-Loeffler-Instituts (SEIDOWSKI et al. 2010), wobei in keiner Studie tote Vögel als Untersuchungsmaterial genutzt wurden. Da das WNV in Amerika mit einem auffälligen Vogelsterben einherging, ist es naheliegend, den Virusnachweis zuerst bei toten Vögeln zu erbringen.

L'épidémie de West-Nile en Amérique du Nord en 2002, qui a touché plus de quarante états aux Etats-Unis, a conduit les Agences de santé à s'interroger sur le risque d'émergence, à l'extérieur de la zone intertropicale, de zoonoses vectorielles. Cette épidémie associée au changement climatique, a bien mis en évidence le rôle central de l'avifaune migratrice dans la diffusion du virus. La biologie des oiseaux, tout particulièrement le phénomène migratoire, permet un transport des virus sur de longues distances et entre espèces très diversifiées. Le Moyen-Orient, qui est situé au carrefour de différents continents, est extrêmement propice à la propagation des virus émergents dans les pays du Nord. La circulation du virus West Nile a été rapportée dans différents pays de la région, tels que l'Egypte, Israël, Liban, Irak, Emirats Arabes Unis et Iran. Saidi et al. (1970) ont montré la présence d'anticorps anti-virus du Nil occidental au sein de la population de la côte caspienne (Nord de l'Iran), des provinces du Khorassan (Nord-Est) et du Khuzestan (Sud-Ouest). Notre étude, conduite dans le cadre d'un programme associant TIMC-IMAG UMR 5525 UJF CNRS VetAgroSup, le Réseau International des Instituts Pasteurs et le Centre National d'Etudes Spatiales, vise: (i) à caractériser la circulation du virus de West-Nile au sein des populations équines d'Iran ; et (ii) et à modéliser l'impact sanitaire de l'environnement et du climat sur la transmission. Les résultats acquis permettent d'apprécier le risque associé à la dissémination spatio-temporelle du virus par les oiseaux migrateurs. Une attention toute particulière est portée à l'étude des déterminants environnementaux et climatiques susceptibles d'accroitre le potentiel de transmission du virus.

Le travail présenté ici s'intéresse au rôle des oiseaux sauvages dans l'épidémiologie du virus West Nile (WN) en Camargue. Des espèces d'oiseaux susceptibles d'intervenir dans les différentes phases de circulation du virus (introduction, amplification, dispersion, émergence) sont identifiées en s'appuyant sur les données bibliographiques relatives à la maladie et sur des critères ornithologiques. Les investigations épidémiologiques effectuées pour quelques unes de ces espèces en 2004 (année épizootique) et en 2005 (année post-épizootique) montrent que le virus WN circule dans la population d'oiseaux de Camargue. Pour les oiseaux migrateurs arrivant d'Afrique au printemps, les dates et lieux de ces contacts restent inconnus. Concernant les oiseaux sédentaires, deux isolats d'une même souche virale ont été obtenus en 2004, réciproquement à partir du cerveau d'une Pie bavarde Pica pica et d'un Moineau domestique Passer domesticus, et totalement séquencés. L'étude phylogénétique de cette souche montre qu'elle appartient au même cluster que celles précédemment isolées en Europe méditerranéenne. Les résultats sérologiques et virologiques chez ces deux espèces d'oiseaux, souvent observées à proximité des écuries, en font des candidates à l'amplification et l'émergence du virus WN chez les chevaux de Camargue. La mise en évidence, en 2005, d'ARN viral dans les fientes d'une Pie bavarde conforte cette hypothèse. Les recherches doivent se poursuivre pour évaluer la part respective des différents oiseaux de Camargue dans la circulation du virus WN en utilisant d'autres approches, comme par exemple l'analyse des repas de sang des moustiques vecteurs, récemment identifiés.

Grundlage der vorliegenden Arbeit ist die Überlegung, dass eine Möglichkeit, die Spezifität der bisher angewendeten Verfahren zur West-Nil-Virus-Diagnostik zu verbessern, in der Anwendung rekombinanter WNV-spezifischer Antigene besteht. Die unter anderem auf bioinformatischen Methoden basierende Identifikation von potenziellen B-Zell-Epitopen und Auswahl entsprechender Sequenzabschnitte richtete sich dabei gezielt auf immunogene Bereiche, die innerhalb der Gruppe der Flaviviren einen ausreichenden Sequenzunterschied zu allen weiteren sequenzverwandten Erregern, zusammengefasst im Japanische Enzephalitis-Serokomplex, boten. Drei ausgewählte Bereiche innerhalb der Strukturproteinsequenz, bezeichnet als prM, Cnat und Cme, sollten mit Hilfe des Expressionssystems Pichia pastoris bzw. Escherichia coli rekombinant exprimiert werden. Nach Erarbeitung optimaler Expressionsbedingungen folgte die affinitätschromatografische Reinigung der im weiteren Verlauf zur Immunisierung von Balb/c-Mäusen eingesetzten Polypeptide. Die gewonnenen Seren der nach verschiedenen Immunisierungsprotokollen geimpften Mäuse wurden im Anschluss immunologisch untersucht. Es zeigte sich, dass die rekombinanten Derivate des Capsid-Proteins eine deutliche Serokonversion hervorriefen. Analysen der mit Cnat und MBP-Cme immunisierten Mausseren wiesen vorhandene peptidspezifische sowie virusspezifische Antikörper nach. Der Einsatz dieser gewonnenen Peptidantigene im indirekten ELISA-Testsystem zur Detektion WNV-spezifischer Antikörper unter Verwendung humaner WNV-IgG-positiver Serumproben zeigte positive Resultate. Im Gegensatz hierzu führte die Immunisierung mit prM lediglich zu einer unspezifischen murinen Antikörperbildung. Die Unterscheidung zwischen WNV-positiven und WNV negativen Humanseren war unter Verwendung des rekombinanten Antigens prM nicht möglich. Im Ergebnis zeigten zwei der drei in dieser Arbeit rekombinant erstellten Strukturproteinabschnitte ihr immunologisches Potenzial in der Generierung muriner WNV spezifischer Antikörper. Zudem konnte mit der Expression der WNV-spezifischen C Protein Antigene ein Beitrag zur Etablierung eines indirekten ELISA-Testsystems zur Detektion WNV-bedingter Humaninfektionen geleistet werden.

Thesis (M.S.) -- Mississippi State University. Department of Geosciences. / Title from title screen. Includes bibliographical references.

Upon viral infection, host cells produce type I interferon (IFN), which activates the JAK-STAT signaling pathway and induces the expression of hundreds of interferon-stimulated genes (ISGs) to establish an antiviral state. In West Nile virus (WNV)-infected cells, the JAK-STAT signaling pathway is blocked by viral proteins. However, the expression of a subset of ISGs, which includes 2¢-5¢-oligoadenylate synthetase 1a (Oas1a), Oas1b, interferon regulatory factor 7 (Irf7), Mx1, and interferon-induced proteins with tetratricopeptide repeats 1 (Ifit1), is still upregulated by an IFN-independent mechanism in WNV-infected mouse embryonic fibroblasts (MEFs). Studies in cells with one or more components of RNA-sensing pathway knocked out showed that the alternative ISG upregulation is activated through RIG-I or MDA5, and the downstream adaptor IPS-1. In cells with IRF3, 5 and 7 knocked out, the alternative ISG upregulation by WNV infection is reduced but not eliminated. As an initial means of discovering the transcription factors involved in this non-canonical ISG upregulation, the critical regulatory regions in the promoters of two representative ISGs, Oas1b and Ifit1, were mapped using a dual luciferase assay system with a NanoLuc luciferase promoter reporter in WNV-infected Ifnar1-/- MEFs. The region from -299 to -28 in the Oas1b promoter, and the region from -192 to -50 in the Ifit1 promoter were identified as being important for upregulating non-canonical gene expression after WNV infection. Fine mapping identified enhancer and repressor sub-regions as well as transcription factor binding sites (TFBSs) putatively involved in the IFN-independent antiviral mechanism. Mutation of one identified TFBS in the ISG promoters reduced Oas1b and Ifit1 promoter activities. In electrophoretic mobility shift assays (EMSAs), a unique band, which was detected in WNV-infected but not in mock-infected Ifnar1-/- MEF nuclear extracts, was not observed when a probe with the identified TFBS mutated was used, suggesting that a unique complex forms at the identified TFBS when it is in the context of the adjacent flanking regions. The unique complex appears to contain NF-κB components and IRF3, IRF5 or IRF7. Our findings provide new insights into the mechanism involved in non-canonical upregulation of ISGs after WNV infection.

West Nile virus (WNV) is a widespread emerging zoonotic neurotropic flavivirus cycling naturally between mosquitos and birds. WNV causes disease in 20% of infections in the most susceptible incidental hosts which are horses and humans. Up to 40% of affected horses and 1- to approximately 50% of affected humans develop neurological signs and/or flaccid paralysis, in some cases fatal or severely debilitating, due to variable encephalitis, meningitis and poliomyelitis. Two predominant genetic lineages exist, 1 and 2, with neurovirulent lineage 1 strains recorded in the northern and western hemispheres, the milder lineage 1 Kunjin strain in Australia, and the lineage 2 strain endemic to southern Africa and Madagascar and considered, until recently, to have mainly mildly pathogenic strains. Since 2002 investigations into South African lineage 2 WNV strains showed that they resulted in severe neurological disease in horses and humans. From 2004 lineage 2 strains were recorded for the first time in southern Europe as a cause of neurological signs and death in birds, and increasingly, in horses and humans. In 2011 the mild lineage 1 Kunjin strain mutated to an equine neurovirulent strain in New South Wales, Australia, and in 2010 the first South African case of lineage 1 WNV was reported from the western Cape in a mare which showed severe neurological signs, abortion and death. Laboratory strains of mice are extremely susceptible to WNV and have been mostly used in experimental studies since the 1937 discovery of the virus in Uganda. In the early 2000s studies in mice showed that field strains of lineage 1 and 2 WNV ranged from mildly pathogenic to highly neurovirulent, however, the associated pathology of the lineage 2 infections was not studied. In the current study, the macroscopic and microscopic pathology of a South African human-neurovirulent field strain of lineage 2 WNV (SPU93/01) and the neurovirulent lineage 1 (NY99/385) strain were investigated and compared in mice used as controls in 2 WNV vaccine studies. The clinical signs, CNS and extra-CNS pathology were indistinguishable between the lineages and some lesions were comparable to those previously reported. Lineage 1 WNV equine pathology has been well described but that of lineage 2 only briefly previously described. The pathology in 6 naturally-occurring fatal lineage 2 WNV-infected horses with severe neurological signs, was investigated and compared with that of the single South African lineage 1 WNV field infection. Diagnoses were confirmed by real-time RT-PCR. Similarities and some slight differences in lesions were found in both mouse and horse studies when compared with lineage 1 pathology cases and with previous reports, and the neurovirulence of the lineage 2 field strains was confirmed. WNV immunohistochemistry (IHC) of all mouse tissues allowed speculation as to pathogenesis of intestinal lesions, but in equine CNS lesions was mostly negative. Ultrastructure of IHC positive cells showed rare WNV particles. In the horse cases rabies, equine herpes virus, and other arboviral co-infections were excluded and similarities and implications of gross lesions of African horsesickness to those often seen in WNV infections were discussed. / Dissertation (MSc)--University of Pretoria, 2014. / gm2014 / Medical Virology / unrestricted

Das West-Nil-Virus (WNV) ist eine Zoonose mit weltweit zunehmender Verbreitung. Natürliches Reservoir dieses Flavivirus sind Vögel, aber auch Säugetiere wie z.B. Menschen können infiziert werden. In einigen Fällen führt eine WNV-Infektion zu schweren neurologischen Erkrankungen. Infolgedessen werden effektive und biologisch sichere Impfstrategien gegen dieses Virus benötigt. Eine Alternative zu herkömmlichen Impfmethoden beschreibt die DNA-Immunisierung. In dieser Arbeit wurde ein potentieller DNA-Impfstoff gegen das WNV hergestellt. Die Immunisierung des DNA-Vektors induzierte starke zelluläre und humorale Immunantworten in Mäusen. Zudem waren die Tiere gegen eine WNV-Infektion geschützt. Zusätzliche Impfungen mit rekombinantem WNV-Protein führten zu einer weiteren Steigerung der Immunogenität des DNA-Impfstoffkandidaten. Des Weiteren sollte der nicht-virale Gentransfer im Allgemeinen optimiert werden. Ein neu entwickeltes Transportsystem für Plasmid-DNA, bestehend aus natürlichen Histonextrakten und Polyethylenimin, resultierte in einer verbesserten Proteinexpression in in vitro transfizierten Zellen und wurde von diesen sehr gut toleriert. Daher wäre diese Strategie auch für zukünftige DNA-Impftechniken denkbar. Der Einfluss von WNV auf die Expression zellulärer miRNAs in Wirtszellen wurde bisher noch nicht untersucht. Dennoch könnten auf diese Weise potentielle molekulare Biomarker für eine frühe WNV-Diagnose identifiziert werden. Mittels Microarray-Technik wurde die Expression zellulärer miRNAs analysiert. Verschiedene miRNA-Spezies waren infolge einer WNV-Infektion leicht herunter- bzw. hochreguliert und stellen mögliche diagnostische Biomarker für das Virus dar.

No description available.