Dissertations / Theses on the topic 'Neurovirulence'

Viral invasion of the central nervous system (CNS) is a significant cause of morbidity and mortlity worldwide, particularly in young children (1). The nervous system presents a challenging site for viruses to access, with multiple physical and immunological barriers that limit pathogen invasion. To invade the CNS, viruses must access cell-surface receptors for binding and entry events. Virus-receptor interactions also govern tropism and often control disease type and severity. For many viruses, the identities of receptors and other cellular determinants of viral tropism remain elusive. Understanding where and how viral capsid components engage neural receptors and the effect of these interactions on tropism and disease may illuminate targets to prevent viral neuroinvasion.

Mammalian orthoreoviruses (reoviruses) provide a highly tractable and well-established system to identify mechanisms of viral entry into the CNS. Reoviruses are non-enveloped particles containing a 10-segmented, double-stranded (ds) RNA genome that replicate well in culture and can be altered via a robust reverse-genetics system (2, 3). While reovirus causes similar age-restricted disease in many young mammals (4-6), most studies employ newborn mice. Following peroral or intracranial inoculation of newborn mice, reovirus displays serotype-specific patterns of tropism in the brain and concomitant disease (Fig. I-1). Serotype 1 (T1) strains infect ependymal cells lining the ventricles of the brain and cause a non-lethal hydrocephalus (7). In contrast, serotype 3 (T3) strains infect specific neuron populations in the CNS and produce a fulminant, and often lethal, encephalitis (8). These differences in tropism and disease have been genetically mapped to the reovirus S1 gene using single-gene reassortant viruses (9). However, viral and host gene sequences that mediate either T1 or T3 tropism have not been defined.

In Chapter I of my dissertation, I introduce key themes about mechanisms of neuroinvasion and the disease consequences of CNS infection. I describe fundamental knowledge and open areas of research pertaining to reovirus infection in the CNS and expand on reovirus-receptor interactions. I conclude Chapter I with a summary of viral oncolytic therapies and highlight strengths and opportunities for improvement of reovirus oncolytics. In Chapter II, I describe the design and implementation of σ1- chimeric reoviruses to identify sequences in the S1 gene that dictate neurotropism and virulence in the CNS. In these studies, I found that homologous sequences at the viriondistal end of the viral attachment protein are responsible for neuron and ependymal cell targeting. In Chapter III, I identify sequences of the NgR1 reovirus receptor that are required for binding and post-binding functions and elucidate the viral ligand for NgR1, which is the σ3 outer-capsid protein, using a combination of genetic, biochemical, and structural approaches. Finally, in Chapter IV, I review conclusions from results presented in Chapters II and III, examine new questions raised by these studies, and discuss future directions of this work. Collectively, my dissertation research has unveiled viral and host sequences that contribute to neural cell targeting and will improve strategies and knowledge to design targeted oncolytic therapies.

Des mutants thermoresistants ont ete isoles a partir de la souche attenuee sabin 1 par des passages successifs a 37. 5#oc, 38. 5#oc et 39. 5#oc. La thermoresistance des quatre mutants etudies a ete confirmee par un test de multiplication a 35#oc et 40#oc. La neurovirulence a ete etudiee chez le singe et s'est averee positive pour tous les mutants (mutants isoles a 37. 5#oc: 2/4 singes paralyses; mutants isoles a 38. 5#oc et 39. 5#oc: 4/4 singes paralyses). Pour tenter d'etablir une correlation entre certaines mutations, la thermoresistance et la virulence, nous avons sequence de facon comparative le genome de la souche sabin 1 et des mutants thermoresistants. Nous avons choisi les regions ou ont ete observees des mutations entre la souche attenuee sabin 1 et la souche virulence mahoney (56 mutations ponctuelles). Nous avons donc sequence 80% du genome de chaque mutant et de la souche parentale sabin 1. Les mutants selectionnes a 39. 5#oc possedent deux mutations dans la region de vp1, une dans celle de vp3, une autre dans le gene de la polymerase virale et deux dans les regions 5 et 3 non codantes. Le mutant selectionne a 38. 5#oc possede la mutation observee dans le gene de la polymerase et les mutations des regions 5 et 3 non codantes. Le mutant selectionne a 37. 5#oc possede les 2 mutations des regions 5 et 3 non codantes. Le neurotropisme du poliovirus a d'autre part ete etudie in vitro dans des cellules de neuroblastome humain. Une infection persistante est etablie et les caracteres phenotypiques des virus persistants different de ceux des virus parentaux. Enfin, nous avons construit et etudie des mutants d'insertion du poliovirus qui portent un nouvel epitope immunogene sur leur capside

Marek's Disease Virus (MDV) induces a wide range of neurological syndromes in susceptible hosts; however, the mechanisms behind the MDV-induced neuropathology are still poorly understood. The immediate-early 14kDa phosphoprotein, pp14, is associated with the neurovirulence phenotype of the virus. Yeast-two-hybrid screening identified the ER-bound transcription regulator, human CREB3 (cAMP Response Element-Binding protein), as an interacting partner of pp14, and fluorescence colocalisation between pp14 and chicken CREB3 (chCREB3) in MDV infected cells suggested an interaction between these proteins. The primary focus of this DPhil project was to further investigate this putative interaction using in vitro studies, with a view to determining if the interaction is linked to the neurovirulence of MDV. This investigation, which employed a combination of biochemical, cellular, and functional assays, found no conclusive evidence in support of the predicted interaction. In addition, this project aimed to gain structural and functional insights into the MDV neurovirulence factor pp14 and the host transcription factor, chCREB3. Biophysical characterisation of recombinant pp14B identifies pp14 as a molten globule. The results reveal the protein, while possessing substantial secondary structure, is largely disordered lacking a stable tertiary structure. Multiple lines of evidence from this study also indicate pp14 is a putative zinc-binding protein. Moreover, phosphorylation analysis of recombinant pp14B, extracted from DF1 cells, by mass spectrometry provides conclusive evidence for the presence of two phosphorylation sites in the shared C-terminal region of pp14 - serines 72 and 76 of pp14B. Structural flexibility, through a lack of a definite ordered tertiary structure, and functional features that can induce structural modifications indicate pp14 might interact with a number of binding partners and therefore could play multiple roles during MDV infection - a strong possibility due to the expression of the protein in all the different stages of virus infection. Furthermore, this thesis presents the crystal structure of the homodimeric chCREB3 bZIP. The chCREB3 bZIP possesses a structured DNA binding region even in the absence of DNA, a feature that could potentially enhance both the DNA-binding specificity and affinity of chCREB3. Significantly, chCREB3 has a covalent intermolecular disulphide bond in the hydrophobic core of the bZIP, which may play a role in promoting stability. Moreover, sequence alignment of bZIP sequences from chicken, human and mouse reveals only members of the CREB3 subfamily possess this cysteine residue, indicating it could act as a redoxsensor. These results indicate members of the CREB3 subfamily, by possessing a putative redox-sensitive cysteine with the capacity to form an intermolecular disulphide bond, may be activated in response to oxidative stress.

O herpesvirus equino tipo 1 (EHV-1) é um importante patógeno que causa doença respiratória, abortamento e desordens neurológicas em equinos. O presente estudo foi realizado visando estabelecer um modelo murino de infecção pelo EHV-1 para investigar a resposta do hospedeiro frente à infecção viral e as alterações neurológicas causadas por esse agente. Camundongos das linhagens BALB/c, BALB/c nude, C3H/HeJ, C57BL/6, C57BL/6 CD4-/- e C57BL/6 CD8-/- foram inoculados por via intranasal com as estirpes brasileiras A4/72, A9/92 e A3/97 do EHV-1. Neste estudo, associou-se a histopatologia, a imunoistoquímica e o método de transcrição reversa seguida pela PCR quantitativa em tempo real para investigar a relação entre a infecção pelo vírus com o desenvolvimento de lesões e a resposta de citocinas pró-inflamatórias no SNC de camundongos das diferentes linhagens. As estirpes brasileiras A4/72 e A9/92 do EHV-1 causaram infecção aguda e letal nas diferentes linhagens de camundongos isogênicos. Os sinais clínicos e neurológicos, tais como perda de peso, pelos arrepiados, postura arqueada, apatia, descarga nasal e ocular, dispnéia, desidratação e sialorréia apareceram entre o 2º e 3º dpi. Essas manifestações foram acompanhadas pelo aumento da sensibilidade a estímulos externos, convulsões, recumbência e morte. O vírus foi consistentemente isolado do SNC, pulmões, fígado, baço e timo de todos os camundongos com sinais neurológicos. As alterações histopatológicas consistiram de leptomeningite, hemorragia focal, ventriculite, degeneração e necrose neuronal, neuronofagia, inflamação não supurativa, gliose multifocal e infiltração perivascular de células polimorfonucleares e mononucleares. A análise imunoistoquímica demonstrou que as estirpes A4/72 e A9/92 do EHV-1 replicaram-se nos neurônicos do bulbo olfatório, cortex cerebral e no hipocampo. Ao contrário, os camundongos inoculados com a estirpe A3/97 do EHV-1 não apresentaram perda de peso ou quaisquer sinais clínicos ou neurológicos; entretanto, o vírus foi isolado dos pulmões no 3º dpi. As estirpes A4/72 e A9/92 do EHV-1 apresentaram tropismo pelo tecido nervoso com capacidade de neuroinvasão e neurovirulência. A estirpe A3/97 do EHV-1 não foi neurovirulenta, apesar de ter sido reisolada do SNC de camundongos BALB/c nude infectados. Detectou-se aumento da expressão de mRNA para TNF-α, IL-6 e CCL2 no SNC dos camundongos infectados pelo EHV-1 com 2 e 3 dpi; entretanto, não houve expressão de mRNA para IFN-γ. Os camundongos com o fundo genético C57BL/6, que apresentam predominantemente resposta do tipo Th1, mostraram níveis mais altos de expressão de mRNA para TNF-α, IL-6 e CCL2, quando comparados com os BALB/c. A gravidade dos sinais observados em camundongos infectados pode ser correlacionada com o pico destas citocinas pró-inflamatórias (TNF-α e IL-6) e da quimiocina CCL2, que são produzidas logo após a infecção viral por células residentes da glia e/ou infiltrativas no SNC. Esses achados indicam que as diferentes linhagens de camundongos isogênicos são susceptíveis a infecção por estirpes neuropatogênicas do EHV-1 e poderiam servir como modelo para o estudo da patogênese e dos mecanismos que contribuem no desenvolvimento da mieloencefalopatia herpética equina.
Equid herpesvirus type 1 (EHV-1) is a major pathogen which causes respiratory disease, abortions and neurological disorders in horses. The present study was carried out to establish a murine model of EHV-1 infection and investigate host response against the virus and neurological disorders caused by this pathogen. BALB/c, BALB/c nude, C3H/HeJ, C57BL/6, C57BL/6 CD4-/- and C57BL/6 CD8-/- mice were intranasally inoculated with EHV-1 A4/72, A9/92 and A3/97 Brazilian strains. In this study, we combined histopathology, immunohistochemistry, and a quantitative real-time RT-PCR method to investigate the relationship between virus infection and the development of lesions and cytokine responses in the CNS of different strains of mice. Intranasal inoculation of EHV-1 A4/72 and A9/92 induced acute and lethal meningoencephalitis in mice. Clinical and neurological signs appeared between the 2nd and 3rd dpi and included weight loss, ruffled fur, a hunched posture, crouching in corners, nasal and ocular discharges, dyspnoea, dehydration and increased salivation. These signs were followed by increased reactivity to external stimulation, seizures, recumbency and death. The virus was consistently recovered from the CNS and visceral organs of all mice with neurological symptoms. Histopathological changes consisted of leptomeningitis, focal hemorrhage, ventriculitis, neuronal degeneration and necrosis, neuronophagia, non-suppurative inflammation, multi-focal gliosis and perivascular infiltration of polymorphonuclear and mononuclear cells. Immunohistochemical examination demonstrated that EHV-1 strains A4/72 and A9/92 replicated in neurons of the olfactory bulb, cortical regions and hippocampus. In contrast, mice inoculated with the EHV-1 strain A3/97 showed neither weight loss nor apparent clinical or neurological signs of the disease; however, the virus was recovered from their lungs at 3 dpi. While EHV-1 strains A4/72 and A9/92 exhibited a high degree of tropism for the CNS with robust neuroinvasiveness and neurovirulence, the EHV-1 strain A3/97 was not neurovirulent despite being detected in the CNS of infected BALB/c nude mice. Increased mRNA levels of TNF-α, IL-6 and CCL2 were detected in the nervous tissue of EHV-1 infected mice at 2 and 3 dpi; however, IFN-γ mRNA was not consistently expressed. Mice with the background C57BL/6, which exhibit predominantly Th1-type responses, showed the highest levels of TNF-α, IL-6 and CCL-2 mRNA in the CNS, when compared to BALB/c mice. The severity of signs observed in infected mice could be correlated with the peak of these proinflammatory cytokines (TNF-α and IL-6) and the chemokine CCL2, which are produced early after viral infection by both cells infiltrating into the CNS from the periphery and/or glial resident cells. These findings indicate that several inbred mouse strains are susceptible to neuopathogenic EHV-1 strains and should be useful models for studying the pathogenesis and mechanisms contributing to equine herpes myeloencephalopathy in horses.

A raiva é uma zoonose quase sempre fatal, que causa milhares de mortes humanas por ano em todo mundo. Essa enfermidade manifesta-se nos mamíferos em duas formas clínicas: furiosa e paralítica. Distinções em relação à evolução, manifestações clínicas, lesões, distribuição e respectiva carga viral no Sistema Nervoso Central (SNC) podem estar relacionadas às características de neuroinvasividade e neuropatogenicidade das diferentes variantes do vírus da raiva (RABV). O objetivo deste projeto foi estabelecer um modelo para o estudo da patogênese da raiva em camundongos inoculados com vírus fixo CVS/31 e variantes de rua do RABV originárias de bovino (variante 3 compatível com isolados de morcegos hematófagos) e de canídeo silvestre (variante compatível com isolados de canídeo silvestre). Para estabelecer o modelo murino, 24 camundongos isogênicos da linhagem BALB/c, de três semanas de idade foram inoculados com 103 DLIC50/0,03mL por via intracerebral (IC) para confirmar a neurovirulência das diferentes variantes do RABV. De modo concomitante, 32 camundongos desta mesma linhagem e faixa etária, foram inoculados com 105 DLIC50/0,03mL pela via coxim plantar (CP) com o intuito de mimetizar a progressão da infecção natural. Para o estudo da patogênese do RABV foram analisados durante um período de trinta dias após a inoculação (DPI) em camundongos pela via CP os seguintes parâmetros: manifestações clínicas, alterações histopatológicas, distribuição antigênica viral pela técnica de imuno-histoquímica (IHQ) e distribuição de RNA viral pela técnica da reação em cadeia da polimerase em tempo real precedida pela transcrição reversa (RT-qPCR) - sistema SYBR Green em diversos segmentos do SNC. Todos os camundongos inoculados com as três amostras do RABV apresentaram sintomas compatíveis com a forma paralítica da doença, tais como: piloereção, perda de peso, postura arqueada, prostração e paresia dos membros posteriores. Apesar de não ser possível observar diferenças de neuropatogenicidade entre as variantes virais, detectaram-se diversidade de virulência entre essas estirpes, demonstrado pelas distinções de período de incubação e taxa de letalidade. Ao analisar os resultados, também foi possível observar diferenças entre a neurovirulência e a neuroinvasividade das diferentes variantes do RABV. Nos camundongos inoculados com CVS/31 pela via CP, no 6º DPI, observaram-se predomínio de manguitos perivasculares na medula espinhal e degeneração neuronal em córtex e intensa distribuição antigênica de RABV no tronco encefálico. Nos camundongos inoculados via CP com amostras do RABV originárias de bovino, no 8º DPI, apresentaram moderada distribuição de manguitos perivasculares na medula e córtex e intensa distribuição antigênica no tálamo. Já nos animais inoculados com a amostra de canídeo silvestre, no 9ºDPI, relataram-se moderada distribuição de manguitos perivasculares no tronco encefálico e moderada distribuição antigênica no córtex. Além disso, foi observado discrepâncias na comparação entre a intensidade e distribuição de marcações antigênicas pela IHQ e inferência semi-quantitativa de distribuição de RNA viral analisada pelo RT-qPCR-sistema SYBR Green no SNC de camundongos
Rabies is a zoonotic disease usually fatal, causing thousands of human deaths each year worldwide. This disease manifests itself in mammals in two clinical forms: furious and paralytic. Distinctions regarding the evolution, clinical manifestations, injuries, distribution and their viral load on the central nervous system (CNS) may be related to the characteristics of neuroinvasiveness and neuropathogenicity of different variants of rabies virus (RABV). The objective of this project was to establish a model to study the pathogenesis of rabies in mice inoculated with fixed virus CVS / 31 and RABV Street variants originating from bovine (variant 3 - compatible with isolates from vampire bats) and wild canid (variant supports isolated from wild canid). To establish the mouse model, 24 mice of the inbred strain BALB/c, three weeks old were inoculated with 103DLIC50/0,03mL intracerebrally (IC) to confirm the neurovirulence of the different variants of RABV. Concomitantly, 32 mice of the same lineage and age were inoculated with 105DLIC50/0,03mL via footpad (CP) in order to mimic the natural progression of the infection. To study the pathogenesis of RABV CP the following parameters were analyzed over a period of thirty days after inoculation (DPI) in mice via CP: clinical, histopathological changes, viral antigen distribution by the technique of immunohistochemistry (IHC) and distribution of RNA by real-time polymerase chain reaction technique preceded by reverse transcription (RT-qPCR) - SYBR Green system in various segments of the CNS. All mice inoculated with the three RABV samples showed symptoms consistent with the paralytic form of the disease, such as ruffled fur, weight loss, hunched, prostration and paresis of the hind limbs. Although it is not possible to observe neuropathogenicity differences between viral variants, the virulence diversity is detected between these strains demonstrated by distinctions incubation period and fatality rate. When analyzing the results, it was also possible to observe differences between neurovirulence and neuroinvasiveness of different variants of RABV. In mice inoculated with CVS/31 via CP on the 6th DPI, there were predominance of perivascular cuffing in spinal cord and neuronal degeneration in cortex and intense antigenic distribution of the RABV in the brainstem. Mice inoculated via CP with RABV samples originating from bovine on the 8thDPI had moderate distribution of perivascular cuffing in the medulla and cortex and intense antigenic distribution in the thalamus. The animals inoculated with the sample of wild canid in 9thDPI reported a moderate perivascular cuffing distribution in the brainstem and moderate antigenic distribution in the cortex. Additionally, discrepancies were observed when comparing the intensity and distribution of antigenic tags by IHC, semi-quantitative inference viral RNA distribution analyzed by RT-qPCR using SYBR Green system in the mouse CNS

O herpesvirus equino tipo 1 (EHV-1) é um importante patógeno que causa doença respiratória, abortamento e desordens neurológicas em equinos. O presente estudo foi realizado visando avaliar a resposta inflamatória causada pelo EHV-1 por meio da análise das manifestações clínicas, alterações histopatológicas e resposta imune do hospedeiro no sistema nervoso central (SNC). Camundongos das linhagens BALB/c (H2d), C57BL/6 (H2b) e C3H/HeJ (H2k) foram inoculados por via intranasal com as estirpes brasileiras A4/72 e A9/92 do EHV-1. Nesse estudo, associou-se a histopatologia, a resposta de citocinas pró-inflamatórias no SNC de camundongos das diferentes linhagens e o método de transcrição reversa seguida pela reação em cadeia da polimerase quantitativa em tempo real (RT-qPCR) para investigar a relação entre a infecção pelo EHV-1 e a resposta inflamatória com o desenvolvimento de lesões. As estirpes brasileiras A4/72 e A9/92 do EHV-1 causaram infecção aguda e letal nas diferentes linhagens de camundongos isogênicos. Os sinais clínicos e neurológicos, tais como perda de peso, pelos arrepiados, postura arqueada, apatia, dispneia, desidratação e sialorreia apareceram entre o 2º e 3º dia pós-infecção (dpi). Essas manifestações foram acompanhadas pelo aumento da sensibilidade a estímulos externos, convulsões, recumbência e morte. As alterações histopatológicas consistiram em necrose neuronal, edema, necrose de liquefação, leptomeningite neutrofílica, manguito perivascular, hemorragia focal, inflamação não supurativa, gliose multifocal e infiltração perivascular de células polimorfonucleares e mononucleares. As características e a extensão das lesões variaram entre as linhagens de camundongos. Animais inoculados com a estirpe A4/72 apresentaram lesões histopatológicas de maior grau de severidade quando comparados com aqueles inoculados com a estirpe A9/92. Observou-se aumento da concentração plasmática de TNF-α, IL-6, CCL2 e IFN-γ nos camundongos infectados pelo EHV-1 no 2º dpi. Detectou-se aumento da concentração plasmática e da expressão de mRNA para TNF-α, IL-6 e CCL2 no SNC dos camundongos infectados pelo EHV-1 no 3º dpi; entretanto, não houve aumento da concentração plasmática nem da expressão de mRNA para IFN-γ no 3º dpi. Evidenciou-se que a estirpe A4/72 do EHV-1 induz uma resposta imune sistêmica mais efetiva, enquanto que o vírus A9/92 culmina em uma resposta imunológica mais efetiva no SNC. Os camundongos com o fundo genético C57BL/6 e BALB/c mostraram níveis mais altos de expressão de mRNA para TNF-α, IL-6 e CCL2, quando comparados com os C3H/HeJ. A gravidade dos sinais clínicos observados em camundongos infectados pode ser correlacionada com o pico dessas citocinas pró-inflamatórias (TNF-α e IL-6) e da quimiocina CCL2, que são produzidas logo após a infecção viral por células residentes da glia e/ou infiltrativas no SNC. Esses achados indicam que as diferentes linhagens de camundongos isogênicos são susceptíveis à infecção por estirpes neuropatogênicas do EHV-1; as diferenças no padrão de alterações histopatológicas mostram que elas dependem do hospedeiro infectado, da estirpe viral e da resposta imunológica; e a supressão do interferon (IFN) tipo 1 sugere ser um mecanismo de escape do EHV-1 frente ao sistema imune. A baixa expressão de IL-6, TNF-α e da quimiocina CCL2 em camundongos C3H/HeJ se explica pela mutação no gene toll-like receptor 4 (TLR-4) existente nessa linhagem de camundongo. Adicionalmente, os camundongos C3H/HeJ apresentaram lesões histopatológicas mais severas no SNC quando comparados com BALB/c e C57BL/6. Sugere-se que o IFN tipo I e o gene TLR-4 apresentam importante papel na patogênese do EHV-1 bem como proteínas do agente viral responsáveis pela supressão do IFN e partículas virais que sejam reconhecidas pelo TLR-4 podem ser alvos para o desenvolvimento de novas abordagens para o tratamento da doença viral e para a eficiência de imunógenos
The equine herpesvirus type 1 (EHV-1) is an important pathogen that causes respiratory disease, abortion and neurological disorders in horses. This study was conducted to evaluate the inflammatory response caused by EHV-1 by the analysis of clinical manifestations, histopathological changes and the host immune response in the central nervous system (CNS). BALB/c (H2d), C57BL/6 (H2b) and C3H/HeJ (H2k) mice were inoculated intranasally with Brazilian EHV-1 strains A4/72 and A9/92. In this study, joined histopathology, the response of proinflammatory cytokines in the CNS of mice of different strains and reverse transcription method followed by quantitative polymerase chain reaction in real time (RT-qPCR) to investigate the relationship between infection by EHV-1 and inflammatory response in the development of lesions. Brazilian strains A4/72 and A9/92 EHV-1 caused acute lethal infection in different strains of inbred mice. Clinical and neurological signs such as weight loss, the bristly hair, hunched posture, apathy, dyspnoea, dehydration and salivary hypersecretion appeared between 2nd and 3rd day after infection (dpi). These events were accompanied by increase in the sensitivity to external stimuli, convulsions, recumbency and death. Histopathological changes were neuronal necrosis, edema, liquefaction necrosis, neutrophilic leptomeningitis, perivascular cuff, focal hemorrhage, non-suppurative inflammation, multifocal gliosis and perivascular infiltration of polymorphonuclear and mononuclear cells. The characteristics and the extent of the injuries varied between strains of mice. Animals inoculated with the A4/72 strain showed histopathological lesions of greater severity when compared with those inoculated with the A9/92 strain. There was an increase in plasma concentrations of TNF-α, IL-6, CCL2 and IFN-γ in mice infected by EHV-1 in 2nd dpi. Plasma concentrations and the expression of mRNA for TNF-α, IL-6 and CCL2 in the CNS of mice infected with EHV-1 at 3rd dpi were increased; however, there was no increase in plasma concentration or expression for the mRNA of IFN-γ at 3rd dpi. It was evident that the EHV-1 strain A4/72 induces a more effective systemic immune response, whereas the A9/92 virus culminates in a more effective immune response in the CNS. The C57BL/6 and BALB/c mice showed higher levels of mRNA expression for TNF-α, IL-6 and CCL2, compared to C3H/HeJ mice. The severity of clinical signs observed in infected mice can be correlated with the peak of these proinflammatory cytokines (TNF-α and IL-6) and CCL2 chemokine, which are then produced after viral infection by resident glial cells and/or infiltrative cells in the CNS. These findings indicate that different strains of inbred mice are susceptible to infection neuropathogenic EHV-1 strains; the differences in the pattern of pathological changes show that they depend on the infected host, the EHV-1 strain and the immune response; and the suppression of interferon (IFN) type I suggested to be an escape mechanism for the EHV-1 against the immune system. The low expression of IL-6, TNF-α and chemokine CCL2 in C3H/HeJ mice can be explained by a mutation in toll-like receptor 4 (TLR-4) gene existing in this mouse strain. Additionally, C3H/HeJ mice exhibited more severe histopathological lesions in the CNS as compared to BALB/c and C57BL/6. It is suggested that type I IFN and TLR-4 gene have important role in the pathogenesis of EHV-1 and viral agent proteins responsible for the suppression of IFN and the viral particles that are recognized by TLR-4 can be targets for the development of new approaches for the treatment of viral disease and the efficiency of immunogens

Abstract Herpes simplex virus type 1 (HSV-1) is a common pathogen with an age-standardized seroprevalence of 52% in Finland. The most common manifestation of HSV-1 infection is labial herpes, but recently HSV-1 has emerged as the most common cause of primary genital herpes in Finnish women. HSV-1 can also lead to severe conditions such as encephalitis. After the primary lytic HSV-1 infection at the epithelia, the progeny viruses infect the innervating sensory neurons. The neuronal infection may lead to a quiescent infection form, called latency. Periodically, the virus may reactivate, which can lead to recurrent infection at the epithelia. During different phases of the viral life cycle the host cells try to restrict the infection. This study set out to investigate the roles of two HSV-1 proteins, γ134.5 and Us3 during different phases of the HSV-1 life cycle. The aim of the first study was to investigate how the deletion of Us3 affected host responses, especially Toll-like Receptor (TLR) signaling, in monocytic U937 cells. TLR3 expression was increased during Us3 deletion virus infections. This also led to increased activation of IRF-3 and increased expression of type I interferons (IFN) and an interferon stimulated protein. This study shows that TLR3 is involved in controlling the HSV-1 infection and that Us3 regulates IRF-3 activation. The second study focused on the role of the γ134.5 protein in HSV-1 latency. Embryonic mouse dorsal root ganglion (DRG) cultures were used as a cell culture model for HSV-1 latency and reactivation. In this model γ134.5 deletion viruses did not reactivate as efficiently as wild-type viruses, even though they replicated well and established latency in the neurons. Stress granules are part of the host response. In the third study, the roles of the innate immunity effectors HSV-1 Us3 and human Z-DNA binding protein 1 (ZBP1) in stress granule formation (SG) were studied. Wild-type HSV-1 efficiently prevented the formation of SGs. The overexpression of ZBP1 resulted in accumulation of smaller but more abundant SGs during oxidative stress. Overexpression of Us3 did not significantly affect the size or number of SGs, but during Us3 deletion virus infection, SG proteins localized to cis-Golgi. This work shows that HSV-1 uses Us3 to evade and modulate host responses and that the γ134.5 protein is required for reactivation in mouse DRG cultures
Tiivistelmä Herpes simplex virus tyyppi 1 (HSV-1) on yleinen taudinaiheuttaja, jonka ikävakioitu seroprevalenssi Suomessa on 52 %. HSV-1 tunnetaan yleisimmin huuliherpeksen aiheuttajana, mutta myös kasvava osuus genitaaliherpeksistä on HSV-1:n aiheuttamia. HSV-1 voi johtaa myös vakaviin ilmentymiin, kuten aivotulehdukseen. Epiteelisolujen infektion tuottamia viruksia siirtyy aluetta hermottaviin tuntohermosoluihin, mikä voi johtaa piilevään infektiomuotoon eli latenssiin. Latentti virus voi ajoittain reaktivoitua, mistä voi seurata uusintainfektio. Isäntäsolu pyrkii rajoittamaan infektiota sen eri vaiheissa. Tämän tutkimuksen tarkoituksena oli selvittää kahden HSV-1:n virulenssiproteiinin, γ134.5:n ja Us3:n, merkitystä HSV-1:n elinkierrossa. Osatyössä I tutkittiin, miten Us3:n poisto vaikuttaa luontaisen immuniteetin vasteisiin, keskittyen etenkin Tollin kaltaisten reseptorien (TLR) signaalivälitykseen U937-monosyyttisoluissa. Us3-poistogeenisillä viruksilla suoritetuissa infektioissa TLR3:n ilmentyminen lisääntyi merkittävästi. Tämä johti myös lisääntyneeseen IRF-3-aktivaatioon sekä tyypin I interferonien ja interferonistimuloituvan proteiinin lisääntyneeseen ilmentymiseen. Tämä osoittaa, että TLR3 osallistuu HSV-1-viruksen tunnistukseen ja että Us3 säätelee IRF-3:n aktivaatiota. Osatyössä II keskityttiin γ134.5-proteiinin merkitykseen HSV-1:n latenssissa. Hiirialkioiden takajuuren hermoganglioita käytettiin soluviljelymallina HSV-1:n latenssin ja reaktivaation tutkimisessa. Tässä mallissa γ134.5-poistogeeniset virukset kasvoivat hyvin ja asettuivat latenteiksi, mutta eivät silti reaktivoituneet kuten luonnonkannan virukset. Stressijyväset ovat osa luontaista immuniteettia. Osatyössä III määritettiin HSV-1:n Us3-proteiinin ja ihmisen Z-DNA:han sitoutuvan proteiini 1:n (ZBP1) merkitystä stressijyvästen muodostumisessa. Luonnonkannan virus kykeni tehokkaasti estämään jyvästen muodostumisen. ZBP1:n yli-ilmentäminen oksidatiivisen stressin aikana johti suureen määrään pienikokoisia stressijyväsiä. Us3:n yli-ilmentäminen ei vaikuttanut stressijyväsiin, kun taas Us3-poistogeenisellä viruksella suoritetuissa infektioissa stressijyväsproteiinit paikantuivat Golgin laitteeseen. Tämä tutkimus osoittaa, että HSV-1 käyttää Us3-proteiinia luontaisten immuunivasteiden muunteluun ja että γ134.5-proteiini on välttämätön reaktivaatiossa hiiren hermoganglioissa

Chez l’homme, la primo-infection par le vírus Herpès simplex de type 1 (HSV-1) a lieu au niveau de la muqueuse oro-pharyngée. Après une phase de réplication, les virions pénètrent les terminaisons axonales et migrent vers le ganglion trijumeau ipsilatéral (TG), puis vers le TG controlatéral. Le virus entre alors dans un état de latence dans les 2 TG. Les réactivations d’HSV-1 dans ces neurones sont responsables des kératites herpétiques (KH), unilatérales et survenant toujours du même côté chez un patient donné.En utilisant un modèle murin oro-oculaire, qui reproduit les aspects essentiels de la maladie chez l’homme, nous avons précédemment démontré que l’inoculation virale latéralisée d’un côté de la bouche entraine une réplication virale dans la lèvre, suivie d’une KH ipsilatérale à l’inoculation 6 jours plus tard. De manière concomitante, le virus se propage aux 2 TG, mais les réactivations ne surviennent que du côté ipsilatéral à l’inoculation. Nous avons également observé qu’après une primo-infection dans une lèvre avec une souche d’HSV-1 non-neurovirulente, les souris étaient protégées contre les signes de la maladie et contre la réactivation d’une souche sauvage pleinement virulente inoculée secondairement dans l’autre lèvre (souche ré-infectante). Afin de comprendre les mécanismes immunitaires en jeu dans cet état de protection, nous avons combiné une analyse en cytométrie en flux multiparamétrique à des tests immunologiques, pour quantifier et définir l’infiltrat immunitaire hématopoïétique et les chémokines inflammatoires au site d’inoculation et dans les TG. Nous avons démontré qu’après une inoculation unique avec la souche sauvage virulente, un infiltrat immun riche en cellules pro-inflammatoires, survenaient de manière retardée dans les lèvres inoculées, et persistaient dans les TG. A l’opposé, l’infiltrat immunitaire était plus précoce dans les tissus réinfectés (après une primo-infection par la souche non neurovirulente), plus riche en cellules de l’immunité adaptative, et associé à des concentrations moindres de chémokines inflammatoires. En outre, cet infiltrat s’estompait plus rapidement, avec une disparition concomitante des chémokines inflammatoires. Ces données permettent de mieux comprendre la nature et la cinétique de la réponse immunitaire anti-HSV-1, et pourront être utiles pour le développement futur de stratégies vaccinales anti-HSV-1
In humans, Herpes simplex virus type 1 (HSV-1), primary infection occurs in the oral mucocutaneous tissues. Virions replicated here penetrate sensitive neuronal axons, migrate to both trigeminal ganglion (TG) where it established a lifelong latency. Reactivations of HSV-1 in the TG neurons induce clinical recurrences in the connected peripheral tissues. This process is involved in herpes simplex keratitis (HSK), a condition that, strikingly, occurs almost exclusively in the same eye for a given patient. Based on an experimental oro-ocular (OO) model of HSV-1 infection, that recapitulates most of these human clinical features, we previously demonstrated that a virus inoculation on one side of the mouth, leads to viral replication in the lip, followed by HSK. Virus concomitantly disseminates to both TG, but reactivation only occurs in the TG ipsilateral to the inoculation site. We also observed that after a primary inoculation with a non-neurovirulent strain of HSV-1 in one lip, mice are protected against both acute phase disease and reactivation after a superinfection with a fully virulent wild-type strain of HSV-1 in the contralateral lip.In order to understand the underlying mechanisms involved in this state of protection, we combined high resolution flow cytometry and bead-based immunoassays, to quantify hematopoietic subsets and inflammatory chemokines in the site of inoculation and in the TG. We demonstrated that after a single inoculation with the wild-type strain, a delayed immune infiltrate, boasting more proinflammatory subsets, occurred in the lip and persisted in the TG. In contrast, the immune infiltrate occurred earlier in the superinfected lip and ipsilateral TG, with less inflammatory chemokines but more adaptive immune subsets. Moreover, cellular infiltrate resolved faster, correlating with nullification of inflammatory chemokines locally. These data show that immune response kinetics influence the development of natural immunity to HSV-1, and can be harnessed to protect against disease and reactivations

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

博士
國防醫學院
生命科學研究所
100
Engineering viruses by inserting microRNA (miRNA) recognition elements (MREs) into the 3’- untranslated region (3’-UTR) of viral RNA can efficiently restrict viral tissue tropism. We used the mosquito-borne Japanese encephalitis virus (JEV) to investigate whether endogenous neuron-specific microRNA-124 (miR-124) could be used to restrict viral neurotropism and, consequently, diminish the neurovirulence of JEV in mice. To recover a neuron-restricted JEV, we inserted 2 copies of a perfectly matched MRE specific to miR-124 into the 3’-UTR to create infectious JEV recombinant RP-124PT (rRP-124PT). The effect of rRP-124PT was attenuated in infected mice as compared with MRE mutant and parental strains, both of which were lethal to challenged mice. Immunization with rRP-124PT appeared to elicit full protective immunity against subsequent JEV lethal challenge. We found neurons of the central nervous system critical targets for infection by JEV, which directly causes lethal encephalitis. The silencing of JEV rRP-124PT in mice by miR-124 illustrates that endogenous miRNA can readily recognize and interact with the 3’-UTR of naturally occurring genomic/mRNAs lacking a polyadenylated tail. Inserting MREs into viral RNA may facilitate further study of flaviviral pathogenesis involving tissue tropism and suggest an additional layer of biosafety for the rational design of safe flavivirus vaccines.

博士
國立成功大學
基礎醫學研究所
95
Enterovirus 71 (EV71), a single positive strand RNA virus that belongs to Picornaviridae, has caused significant morbidity and mortality worldwide since it was first described in 1969 in the United States. EV71 infections usually cause epidemic HFMD and occasionally have been associated with aseptic meningitis, encephalitis and polio-like illness. The outbreak of EV71 in Taiwan in 1998 killed 78 children. It has been suggested that EV71 may become the most important neurotropic enterovirus after the eradication of poliovirus. One of the major barriers to study the pathogenesis of EV71 and vaccine development is the lack of a suitable animal model. In an attempt to establish a murine model of EV71 infection, we found that mouse-adapted EV71 strains, which were isolated from the brain of ICR neonatal mice infected intraperitoneally at the age of day 1, were more virulent than the parental strain. Furthermore, MP4 (the fourth passage of mouse-adapted EV71 strain), in contrast to its parental strain EV71/4643, could orally infect and replicate in the central nervous system of neonatal mice, indicating an increase in neuroinvasiveness of the virus after adaptation. In vitro mouse-adapted EV71 strains exhibited a more rapid growth rate, bigger plaque size, lower temperature-sensitivity and more resistant to heat treatment at 45�aC and acidic condition than parental strain 4643. Oral inoculation of MP4 into 7-day-old mice caused encephalitis, flaccid paralysis and death of mice at 7-9 days post infection. Pathological examination revealed inflammatory cell infiltration and neuron apoptosis in the brain and spinal cord, massive necrosis in the limb muscles and severe lymphocytes depletion in the lymphoid organs. After oral inoculation, there was an early and transient virus replication in the intestines, whereas the spinal cord, brain and muscle became the sites of viral replication during the late phase of the infection. Protection against EV71 challenge was demonstrated following administration of hyper-immune serum after inoculation of the virus. Nucleotide (nt) sequence analysis of the mouse-adapted EV71 strains genome revealed there were thirty-one nucleotide changes after four in vivo passages, resulting in five amino acid (aa) substitutions. Interestingly, MP1, the first passage of the virus, had 29 nt and 4 aa changes with increased resistant phenotypes (rct marker, thermostability and acid resistance) but not in vivo toxicity. Five additional nt changes and three aa substitutions (1 in VP1, E710Q, and 2 in 2A, V964M and A1009V) were occurred in the transition of MP1 to MP2. MP2 and MP3, the second and third passages of parental strain 4643 in vivo, were strongly acid resistance and more thermostable and lethal to 1-day-old mice than the 4643 with a LD50 of 1 log-fold decrease. MP4, the virus strain after the forth passage of 4643, was highly toxic to 1-day-old mice (2 log-fold decrease in LD50) and acquired the ability to invade the central nervous system after oral delivery. The MP4 had a total of 31 nt and 5 aa different than those of 4643, and only one aa different between MP1 and MP4 which located in the VP1 region (E710Q). Studies with the luciferase reporter plasmid demonstrated that the IRES activity of MP4 was higher than parental strain 4643 in both RD cells and SK-N-SH cells. Furthermore, the full-length infectious cDNA of 4643 and MP4 were constructed and used to generate the chimeric viruses. The 4M-1, a cDNA clone after replacing the 5’ terminal 964 nucleotides of MP4 genome with the corresponding region of the 4643 strain, produced MP4-like plaques in RD cells and showed neurovirulence similar to that of MP4 in neonatal mice, but had a kinetic of replication in RD cells similar to 4643. We concluded that, unlike poliovirus, the 5’ UTR of EV71 may be important for viral replication and that its function alone is not sufficient for large plaque phenotype and mouse virulence. We believe that both mouse-adapted EV71 strains and murine model will provide excellent frameworks for examining the pathogenesis of EV71, verifying the virulence determinants of EV71, and developing vaccines and anti-EV71 drugs.

碩士
國立陽明大學
醫學生物技術暨檢驗學系暨研究所
97
Enterovirus 71 (EV71), a member of Picornaviridae family, can lead to severe neurological complications, and has caused several large outbreaks in Taiwan since 1998. The 5’ untranslated region (5’-UTR) of viruses is highly structured, containing the internal ribosomal entry site (IRES) that is critical for translation initiation. The efficiency of IRES-dependent translation is concerned with the infectivity of viruses, but whether the IRES is concerned with neurotropism or not is still controversial. On the other hand, the IRES-dependent translation initiation needs noncanonical factors called IRES trans-acting factors (ITAFs). Nowadays, the understanding of EV71 ITAFs is still limited. In this study, we used a dual-luciferase reporter system that harbored IRES from EMCV, EV71 BrCr strain, EV71 B5 strain Taiwan isolate, Sabin type 3 vaccine strain, Sabin type 3 revertant strain, and CA16. By analyzing the IRES activities in neural and non-neural cells, we found that different viruses showed distinct IRES activities, yet each IRES activity is comparable in the two origins of cells. Accordingly, we suggested that the neurotropism of human enterovirus is not governed by viral IRES. Additionally, we investigated La, PTB, PCBP2, and Unr, the known ITAFs of poliovirus, on their roles in interacting with EV71 IRES. Utilizing RNA interference strategy to knock down these proteins in HeLa cells, we showed that knock down of Unr, but not other ITAFs, could significantly decrease EV71 IRES activity. The decreased level of Unr also led to reduced viral protein expression and viral particle production. We revealed that Unr, among the ITAFs investigated, played the most significant role in controlling EV71 IRES.

Masernvirus (MV) ist ein negativ-strängiges RNA-Virus, das im Menschen und im Nagermodell zu akuten und subakuten Enzephalitiden führen kann. Es wurde beschrieben, dass bestimmte Antikörperescape-Mutanten des MV neurovirulent, andere nicht neurovirulent sind (Liebert et al., 1994). Mit Hilfe von rekombinanten Masernviren, konnte ich diejenigen Aminosäuren charakterisieren, die einerseits für die Bindung monoklonaler, neutralisierender anti-MV-H-Antikörper (K29, K71, Nc32 und L77) und andererseits für die Neurovirulenz verantwortlich sind. Bei den rekombinanten MV wurde das von Duprex et al. (1999) als Neurovirulenz vermittelnd beschriebene H-Gen des nagerhirnadaptierten neurovirulenten CAM/RB-Stammes in das Grundgerüst des nicht neurovirulenten Edtag (molekularer Klon des Vakzinestammes Edm) kloniert. Über gerichtete Mutagenese wurden die jeweiligen Mutationen in dieses CAM/RB H-Gen eingefügt. Mittels der FACS-Analyse konnten die Aminosäureänderungen identifiziert werden, die für die Bindung der jeweiligen Antikörper verantwortlich sind. Sie befinden sich nach einem Strukturmodell der H-Proteine (Langedijk et al., 1997) im Membran-distalen Teil, den so genannten Propellern. Im Einzelnen sind folgende Aminosäureänderungen im Hämagglutinin-Protein für den Escape verantwortlich: L77 – 377 Arg -> Gln und 378 Met -> Lys; Nc32 – 388 Gly -> Ser; K71 – 492 Glu -> Lys und 550 Ser -> Pro; K29 – 535 Glu -> Gly. Es konnte ferner gezeigt werden, dass die beiden Aminosäureveränderungen an den Positionen 195 und 200 gemeinsam für die Neurovirulenz verantwortlich sind und nicht assoziiert sind mit den Mutationen für den Antikörperescape. Der Aminosäureaustausch an Position 200 bei neurovirulenten Viren führt zum Verlust einer benutzten Glykosylierungsstelle. Diese Mutation ist jedoch nicht alleine für das unterschiedliche Neurovirulenzverhalten der Viren verantwortlich, sondern es muss gleichzeitig der Austausch an Position 195 vorhanden sein, der eine positive Ladung im H-Molekül entfernt. Diese beiden Mutationen sind nach dem Strukturmodell nach Langedijk im Stamm2-Bereich angesiedelt. Sind im H-Protein an Stelle 195 und 200 die Aminosäuren Gly und Ser vorhanden, so findet im Gehirn neugeborener Lewis-Ratten eine verstärkte Virusvermehrung und Ausbreitung statt, die die akute Enzephalitis mit Expression typischer proinflammatorischer Zytokine zur Folge hat. Werden an Stelle 195 und 200 die Aminosäuren Arg und Asn exprimiert, so ist der Verlauf der Infektion inapparent. In dieser Arbeit wurde auch ein Zellkultursystem gemischter Hirnzellen neugeborener Lewis-Ratten etabliert, das die Unterschiede der Virusausbreitung in vivo reflektiert und mit dem weitere Untersuchungen zum Mechanismus der Neurovirulenz durchgeführt werden könnten. Anhand der durchgeführten Untersuchungen mit Ratten des CD46 transgenen Lewis-Modells konnte gezeigt werden, dass die Anwesenheit des Rezeptors CD46 das Virulenzverhalten der getesteten Viren nicht beeinflusst. Weder mit dem Vakzinestamm Edm noch mit einem nicht an Nager adaptierten Wildtypstamm, konnte nach intracerebraler Injektion eine akute Enzephalitis induziert werden. Die Untersuchungen zeigen, dass die Neurovirulenz des an Nager-adaptierte MV-Stammes CAM/RB essentiell von den Aminosäuren Gly und Ser an Position 195 und 200 im H-Protein abhängt und nicht durch die transgene Expression zellulärer Rezeptoren für MV vermittelt werden kann
Measles virus (MV) is a negative stranded RNA-virus, which may lead to acute and subacute encephalitis in men and experimentally also in rodents. It has been described that certain antibody escape mutants of MV are neurovirulent, whereas others are non-virulent (Liebert et al., 1994). Here I determined with the help of recombinant MV the amino acids which are responsible for the binding of neutralizing monoclonal anti MV-H-antibodies (K29, K71, Nc32 and L77) or for neurovirulence of MV. The H-gene of the rodent brain adapted strain CAM/RB which was described for determining neurovirulence by Duprex et al. (1999) was introduced into the non-neurovirulent backbone of Edtag, which is the molecular clone of the vaccine strain Edm. The respective mutations were introduced by site directed mutagenesis. In FACS-analysis I could determine the amino acid changes which are responsible for the binding of the anti H-antibodies. In a structural model for MV-H (Langedijk et al., 1997) this amino acids reside in the membrane distal part of the molecule - the so called propeller. The following amino acid changes in the hemagglutinin protein are responsible for the antibody escape: L77 – 377 Arg -> Gln und 378 Met -> Lys; Nc32 – 388 Gly -> Ser; K71 – 492 Glu -> Lys und 550 Ser -> Pro; K29 – 535 Glu -> Gly. In addition I found that the combined amino acid changes at positions 195 and 200 are responsible for neurovirulence but are not associated with the antibody escape. The amino acid change at position 200 leads to the loss of a used glycosylation site in neurovirulent strains. The mutation at position 200 is not alone responsible for the neurovirulence but requires the second associated mutation at position 195, which deletes an additional positive charge in the H-protein. These two mutations which are responsible for the neurovirulence reside in the stem2 region of the structural model according to Langedijk. If in the H-protein the amino acids at position 195 and 200 are Gly and Ser, the virus multiplication and spread is enhanced in the brain of newborn Lewis-rats and causes an acute encephalitis with expression of typical proinflammatory cytokines. If at position 195 and 200 the amino acids Arg and Asn are present, the infection stays inapparent. I could also establish a cell culture system of mixed primary rat brain cells, which reflects the difference in the viral spread in vivo and which may be to used further to investigate the mechanisms responsible for neurovirulence. Results obtained with CD46 transgenic Lewis-rats showed that the presence of the MV receptor CD46 does not influence the virulence of the tested strains. Neither the vaccine strain nor a wildtype strain not adapted to rodents could induce acute encephalitis after intracerebral injection. These findings suggest that the neurovirulence of the rodent-brain adapted MV-strain CAM/RB depends essentially on amino acids Gly and Ser at positions 195 and 200 in the H-protein, and cannot be mediated by the transgenic expression of cellular receptors for MV