Dissertations / Theses on the topic 'Pneumovirus'

Orientador: Clarice Weis Arns<br>Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia<br>Made available in DSpace on 2018-07-26T23:35:22Z (GMT). No. of bitstreams: 1 Monteiro_MariaCeliaGarciaBezzan_M.pdf: 3780766 bytes, checksum: 2e7830137747659681f5ac3581919f0c (MD5) Previous issue date: 2000<br>Mestrado

Orientador: Clarice Weis Arns<br>Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia<br>Made available in DSpace on 2018-08-04T11:34:54Z (GMT). No. of bitstreams: 1 D'Arce_ReginaCeliaFreitas_D.pdf: 5035070 bytes, checksum: 70450102e2aa6208d35d5a038918c516 (MD5) Previous issue date: 2004<br>Resumo: O presente estudo teve como objetivo o desenvolvimento de técnicas moleculares diferenciais para o diagnóstico do Vírus da Bronquite Infecciosa (VBI), responsável por infecções respiratória e urogenital agudas, altamente contagiosas em frangos e galinhas, e do Pneumovírus Aviário (PVA), agente etiológico da Rinotraqueíte dos Perus e associado à Síndrome da Cabeça Inchada (SCI) em galinhas. Sorotipos do VBI e subtipos do PV A foram cultivados em ovos embrionados e em células CER (Chicken Embryo Related), respectivamente. Foram adaptadas as técnicas de extração do RNA viral, RT -PCR, nested-PCR subtipo-especmco. para o PVA e RT -PCR-RFLP para o VBI a partir dos vírus cultivados, sendo testadas posteriormente em material coletado de aves infectadas experimentalmente. Tanto o PVA como o VBI foram detectados por RT-PCR e/ou nested-PCR nos grupos infectados com os agentes isoladamente ou em co-infecção, principalmente nos dias 3 e 6 pós-inoculação. As técnicas diagnósticas desenvolvidas foram então utilizadas na detecção viral de materiais clínicos, obtidos de plantéis avícolas com histórico de problemas respiratórios e sorologia positiva para os agentes estudados. Para o PVA obteve-se a detecção de oito vírus de campo, todos pertencentes ao subtipo A, donde foi possível o isolamento de quatro amostras. No caso do VBI foram detectadas três amostras de campo, todos diferenciados da amostra vacinal por RFLP, dos quais isolou-se duas amostras em ovos embrionados. Amostras positivas tiveram seus fragmentos de PCR seqüenciados, confirmando a classificação obtida através da nested-PCR para o PVA e do RFLP para o VBI. A análise das seqüências do VBI mostrou que os isolados de campo diferem de todos os sorotipos descritos até o momento. As técnicas molecu1ares otimizadas nesse estudo confinaram sua capacidade em diferenciar os subtipos A e B do PV A, bem como as variantes detectadas de VBI de campo e o sorotipo vacinal, demonstrando ser adequadas alternativas diagnósticas e valiosas ferramentas para estudos de epidemiologia molecular<br>Abstract: The aim of this study was the development of molecu1ar techniques able to differentiate Infectious Bronchitis Virus (IBV), responsible for high 'contagious acute respiratory and renal infections in chickens, and Avian Pneumovirus (APV), causal agent of Turkey Rhinotracheitis and associated to Swollen head Syndrome (SHS) in chickens. APV and IBV strains, representatives of subtypes A and B and serotype Massachussets, were multiplied in Chicken Embryo Related (CER) cell line and embrionated eggs, respectively. The techniques of RNA extraction, RT -PCR, APV subtype-specific nested-PCR and RT -PCR-RFLP for IBV diagnosis were adapted, and then tested in experimentally infected chickens. Both viruses were detected by RT -PCR and/or nested-PCR in the groups infected with the agents separately or in co-infection, mainly at 3rd end 6th day post infection. Moreover, these techniques were utilised in clinical field samples from chicken and turkey flocks with respiratory problems and positive APV and IBV serology. Eight APV were detected, all of them classified as subtype A, where four samples were isolated. In the case of IBV, three field samples were detected, originating two viral isolation, all different from vaccine serotype by RFLP. The PCR products were sequenced, confirming the APV nested-PCR and IBV RFLP classification. IBV sequence analysis showed that the Brazilian field samples detected in this study are distinct from all serotype described 50 far. These optimised techniques could be useful to direct differentiation of APV subtypes A and B, and IBV field and vaccine strains, proving to be a valuable molecular epidemiological tool<br>Doutorado<br>Microbiologia<br>Doutor em Genetica e Biologia Molecular

Orientador: Helio Jose Montassier<br>Resumo: As infecções do trato respiratório de aves têm-se constituído em problemas crescentes e com marcantes consequências negativas sobre a produção avícola em várias partes do mundo, notadamente onde a avicultura é mais desenvolvida como no Brasil. Dentre essas enfermidades, destacam-se as micoplasmoses aviárias e a pneumovirose aviária, que, apesar de suas relevâncias em sanidade avícola, não têm sido investigadas de forma sistematizada no Brasil, em especial no que concerne à interação entre esses agentes ou a ocorrência de co-infecção em frangos de corte. O presente estudo investigou a ocorrência de infecção por Mycoplasma gallisepticum (MG) e pelos subtipos A ou B de Metapneumovírus aviário (AMPV) em frangos de corte de plantéis avícolas comerciais mantidos em granjas mais tecnificadas localizadas nas regiões Sudeste e Centro-Oeste do Brasil. Para tanto, as técnicas de PCR e de RT-Nested-PCR foram usadas na detecção e/ou identificação, respectivamente de MG e AMPV em amostras de suabes nasais e traqueais colhidos de 87 lotes de frangos de corte com problemas respiratórios e oriundos de 15 granjas de produção comercial de frangos de corte. Dos lotes amostrados, dois deles em um total de 87 (2,3%) e de uma única granja da região Sudeste, mostraram-se positivos para MG, enquanto que nenhum dos lotes investigados revelou-se positivo para AMPV. A baixa ou nenhuma incidência desses agentes pode ser explicada pela utilização de medidas cada vez mais efetivas para o controle sanitário... (Resumo completo, clicar acesso eletrônico abaixo)<br>Abstract: Respiratory infections of poultry may be an increasing and negative problem with poultry production in several parts of the world, especially when poultry farming is more widely used than in Brazil. These diseases include avian mycoplasmosis and avian pneumovirosis, which, despite their relevance in poultry health, have not been systematically investigated in Brazil, especially with regard to the interaction between these agents or the occurrence of co-infection in broilers. The present study investigated the occurrence of infection by Mycoplasma gallisepticum (MG) and A or B subtypes of avian Metapneumovirus (AMPV) in broiler commercial poultry farms located in the Southeast and Center-West regions of Brazil. For this, PCR and RT-Nested-PCR techniques were used in the detection and / or identification, respectively of MG and AMPV in nasal and tracheal swab samples collected from 87 lots of broilers with respiratory problems and from 15 commercial production of broilers. Of the sampled lots, two of them in 87 (2.3%) were positive for MG, whereas none of the lots were positive for AMPV. The low or no incidence of these agents can be explained by the use of increasingly effective measures for the sanitary control of these agents in the commercial farms of sampled broilers. Our findings also suggest, however, that other bacterial and viral infectious agents not investigated in this study may be involved in the etiology of the respiratory problems of these birds, since those that... (Complete abstract click electronic access below)<br>Mestre

Le métapneumovirus humain (hMPV) est un virus responsable d'infections aiguës des voies respiratoires telles que des bronchiolites, des bronchites ou des pneumonies, principalement chez les populations à risques que sont les jeunes enfants de moins de 5 ans, ainsi que les personnes âgées ou immunodéprimées. Découvert en 2001, ce virus et sa pathogénèse ne restent encore aujourd'hui que partiellement caractérisés. De ce fait et malgré les besoins, il n'y a aucun vaccin ou traitement thérapeutique spécifique et efficace contre le HMPV disponible sur le marché. Dans ce contexte, mon projet de thèse s'est articulé autour de deux axes principaux : (i) L'étude de la protéine de fusion F du virus hMPV, protéine majeure antigénique de surface et responsable de l'entrée du virus dans la cellule cible. Elle a pour particularité d'induire de manière autonome la fusion membranaire in vitro et d'être associée à des effets cytopathiques variable selon les souches virales. De par son rôle clé pour le virus hMPV, la protéine F a déjà fait l'objet de plusieurs études structurales et fonctionnelles mais les déterminants de cette activité fusogénique ne sont pas encore entièrement caractérisés. Nous nous sommes donc intéressés à l'identification de déterminants du phénotype viral hyperfusogénique, localisés dans les domaines heptad repeats de la protéine F du hMPV. (ii) L'atténuation de deux souches virales cliniques (CAN98-75 et C-85473) par délétion de gènes accessoires dans le but de développer des candidats vaccinaux adaptés aux enfants en bas âge. Différents virus ont été générés par génétique inverse et les délétions des gènes accessoires SH et G dans les deux fonds génétiques viraux ont été étudiées pour leur impact sur l'infectivité, la réplication et la pathogénèse virale in vitro et in vivo ainsi que leur contribution pour le développement de virus atténués candidats vaccinaux<br>Human metapneumovirus (hMPV) is a major pathogen responsible of acute respiratory tract infections, such as bronchiolitis or pneumonia, affecting especially infants, under five years old, elderly individuals and immunocompromised adults. Identified since 2001, this virus and its pathogenesis still remain largely unknown and no licensed vaccines or specific antivirals against hMPV are currently available. In this context, my research project was built over two main subjects: (i) The study of the fusion F glycoprotein which is the major antigenic protein of hMPV and is responsible of viral entry into host cell. By its crucial role for the virus, the F protein has already been characterized in several structural and/or functional studies. Thus, it has been described that the hMPV F protein induces membrane fusion autonomously, resulting in variable cytopathic effects in vitro, in a strain-dependent manner. However, as the determinants of the hMPV fusogenic activity are not well characterized yet, we focused on identification of some of these, located in heptad repeats domains of the protein. (ii) The evaluation of hMPV SH and G gene deletion for viral attenuation. Liveattenuated hMPV vaccine candidates for infants’ immunization has been constructed thank to this deletion approach at the beginning of hMPV vaccine development efforts. Despite encouraging results, these candidates have not been further characterized and the importance of the viral background has not been evaluated

Orientador: Clarice Weis Arns<br>Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia<br>Made available in DSpace on 2018-07-23T20:36:49Z (GMT). No. of bitstreams: 1 Dani_MariaAngelaGomesdeCastro_M.pdf: 3584513 bytes, checksum: 44f0c60386e93a6a79298e7ba5df2fba (MD5) Previous issue date: 1998<br>Resumo: Pneumovírus aviário (PVA) causa infecção aguda no trato respiratório em perus (rinotraqueítedos perus) e galinhas (síndrome da cabeça inchada) com início abrupto e disseminação rápida. Neste estudo, foi padronizada a reação de RT-PCR quimioluminescente para detecção de um transcrito do gene F do PVA em dois isolados europeus e dois isolados brasileiros. Foram realizadas PCRs de diluição limitante utilizando-se RT-PCR quimioluminescente e "nested PCR" (nPCR) para a comparação quanto à sensibilidade e especificidade entre as metodologias na detecção de PVA. A sensibilidade e especificidade da RT-PCR quimioluminescente foram semelhantes às da nPCR e 100 vezes mais sensível que uma única PCR. O seqüênciamento dos produtos de 175 pb do gene F revelaram 100% de homologia com seqüências depositadas no banco de dados. Em seguida foi realizada a caracterização dos isolados brasileiros através de RT-PCR, análise com enzimas de restrição e seqüênciamento automático de um fragmento do gene G. Os produtos de 600 pb correspondentes aos primeiros 600 nucleotídeos do gene G dos isolados SHS BR 119 e SHS BR 121 revelaram 99% de similaridade com 3 isolados do subgrupo A e 87% de similaridade com isolados do subgrupo B depositadas no banco de dados<br>Abstract: Avian Pneumovirus (APV) causes an acute respiratory tract infection both in turkeys (turkey rhinotracheitis) and chicken (swollen head syndrome) with sudden onset and rapid spread through the flocks. In this study, immunochemiluminescent Southern blot RT-PCR assay was employed to detect a F gene transcript of the avian pneumovirus (APV) in two European turkey isolates and two Brazilian chicken isolates. Limiting dilution PCR was performed to compare the sensitivity of immunochemiluminescent Southern blot assay and nested PCR (nPCR) assay. The sensitivity and specificity of immunochemiluminescent Southem blot RT-PCR assay were comparable to that of nPCR, and at least 100 fold more sensitive than a single PCR amplification. Sequence analysis of the 175 bp product ofthe F gene revealed 100% identity with earlier reported PVA sequences. We characterized the Brazilian isolates using restriction analysis and sequencing of a 600 bp fragment of the G gene and conclude that the isolates belong to the APV subgroup A<br>Mestrado<br>Microbiologia<br>Mestre em Ciências Biológicas

Les métapneumovirus aviaires (AMPV) sont responsables d’infections aiguës des voies respiratoires et du tractus génital chez les volailles d’élevage – dont notamment les dindes, poulets et canards – ayant d’importantes conséquences pour l’industrie avicole. A ce jour, quatre sous-groupes (A, B, C et D) et deux lignées d’AMPV-C (Us et Fr) ont été établis selon leurs différences génétiques et antigéniques. Cependant leurs spectres d’hôtes n’ont encore jamais été clairement élucidés. Pour résoudre ce problème, la première partie de cette thèse a consisté à étudier le spectre d’hôte, la pathogénicité comparée et la transmission horizontale des AMPV chez les dindes, poulets et canards. Les résultats de cette étude révèlent que les AMPV-A, B, Us-C et D sont mieux adaptés aux galliformes (dindes et poulets) et que le Fr-AMPV-C est mieux adapté aux palmipèdes (canards). Fait intéressant, des AMPV-C des deux lignées ont été réisolés chez des hôtes pouvant être décrits comme « non conventionnels », à savoir Fr-AMPV-C chez des galliformes et Us-AMPV-C chez des poulets, avec des éléments suggérant que l’expression ou l’intégrité du gène SH ont été modifiés. Pour tester le rôle de SH dans le spectre d’hôte, la seconde partie de ma thèse a consisté à développer un système de génétique inverse pour le Fr-AMPV-C permettant la régénération de virus chimérique dont la protéine SH a été échangé avec celle d’un Us-AMPV-C. Ces virus recombinants ont été testés chez des dindes et canards EOPS en conditions expérimentales. Le virus chimérique n’a pas montré de modification de son phénotype en comparaison avec le virus parental, ce qui suggère que les différences de tropisme d’hôte des différentes lignées d’AMPV-C ne sont pas liées au type de protéine SH. Cependant, cela doit être confirmé en régénérant un virus Us-AMPV-C chimérique qui exprime la protéine SH de Fr-AMPV-C et en effectuant les mêmes essais expérimentaux sur des dindes et canards EOPS<br>Avian metapneumoviruses (AMPV) cause respiratory and genital disorders in poultry, most notably, turkeys, chickens and ducks and are a major economic concern for the industry. To date, four subgroups of AMPV (A, B, C and D) and two genetic lineages of AMPV-C (Us and Fr) have been identified based on antigenic and genetic differences. However, their host tropism is not clearly defined. To address this issue the first part of the thesis focused on the host spectrum of the different subgroups and lineages of AMPV in turkeys, chickens and ducks, in respect to pathogenicity and horizontal transmission of the viruses. Globally, the results showed that AMPV-A, B, Us-C and D were well adapted to Galliformes (turkeys and chickens) and that the Fr-AMPV-C virus was well adapted to ducks. Interestingly, infectious AMPV-C viruses of both lineages were re-isolated from what could be considered as “non-conventional” hosts i.e. galliformes for Fr-AMPV-C and chickens for Us-AMPV-C with indications of genome modifications that altered the expression or the integrity of the SH gene. To test a hypothesis that SH plays a role in host tropism, the second part of the thesis consisted in the development of a reverse genetic system for Fr-AMPV-C from which recombinant Fr-AMPV-C viruses with SH proteins derived from Us-AMPV-C’s were rescued. These viruses were then tested in SPF ducks and Turkeys under experimental conditions. Recombinant viruses did not demonstrate any notable alteration in host tropism in respect to the parental viruses. Thus it would be reasonable to assume that host tropism differences among AMPV Cs are not linked to the type of SH protein however, this needs to be confirmed by rescuing recombinant Us-AMPV-C viruses that express the SH protein of Fr-AMPV-C’s and performing the same experimental trials in SPF ducks and turkeys

Members of the Pneumoviridae virus family are responsible for severe respiratory tract disease in their hosts. Human respiratory syncytial virus (hRSV) is responsible for over 200,000 deaths worldwide each year and bovine respiratory syncytial virus (bRSV) causes major economic loss to the cattle industry worldwide. The current model for all nonsegmented negative-sense single stranded RNA virus gene expression, is that mRNA is generated in a polar gradient, with decreasing levels of mRNA transcribed from genes further along the genome from the 3 ́ end. With the exception of translation of ORF-2 located on the bicistronic M2 mRNA, translation of Pneumoviridae mRNAs is thought to be regulated through the levels of mRNA abundance. Translation of M2 ORF-2 has been characterised as being regulated by the non-canonical mechanism of coupled translation termination/initiation in pneumonia virus of mice (PVM), hRSV and avian metapneumovirus (APV). This mechanism is reliant on a proportion of the elongating ribosome translating the upstream M2 ORF-1, terminating and reinitiating translation of M2 ORF-2. Although the initiation site for M2 ORF-2 is similar in bRSV to other members of this family that use the mechanism of coupled translation, the mechanism has not been characterised. Using the technique of ribosomal profiling to analyse steady state viral mRNA abundance and viral translation in both hRSV and bRSV-infected cells, it was observed that for certain viral mRNAs, levels of mRNA abundance did not follow the standard polar transcription model. This was characterised by an increase in the levels of mRNA abundance between the mRNA’s respective gene and its upstream neighbour. The increase was observed in the same group of mRNAs in both viruses suggesting that factors other than the transcription polar gradient influence levels of viral mRNA abundance. It was also observed that levels of proportional translation did not match the respective proportional levels of mRNA abundance for certain viral mRNAs in both viruses. This would suggest that translation of viral genomes is not primarily controlled by mRNA abundance and instead other translational regulatory factors influence levels of translation. The mechanism of bRSV M2 ORF-2 translation was also characterised using reporter plasmids assays. It was identified that the mechanism of initiation of translation of M2 ORF2 used, was not that of coupled translation termination/initiation used by other members of this family. Instead it was observed that translation of M2 ORF-2 used an internal initiation mechanism located inside M2 ORF-1 to initiate translation. The mechanism of coupled translation termination/initiation used for translation of PVM M2 ORF-2 was also further characterised. It was observed that translation of M2 ORF-2 was reliant on upstream sequence in the M2 ORF-1 sequence. A predicted mRNA secondary structure was identified in this region and when disrupted, inhibited translation of M2 ORF2. This was similar to the mechanism of coupled translation used in hRSV, suggesting that the mechanism used by this family is reliant on a mRNA secondary structure located upstream of the initiation site.

The work described in this thesis identifies some properties of the major polypeptides of pneumonia virus of mice (PVM) and of turkey rhinotracheitis (TRT) virus. The PVM glycoproteins have been studied in particular detail while the results obtained with TRT virus provide a preliminary description of the polypeptides of this virus. Twelve major PVM specific polypeptides designated L, G1, G2, F1, N, 39K, 35K, M, 20K, 19K, 16K and 12K were identified. In addition PVM specific polypeptides designated 25K, 24K, 23K, 18K and 17K were sometimes detected. Monoclonal antibodies directed against the G1/G2, 39K and M polypeptides were produced. The a~ility of a monoclonal antibody to precipitate G1 and G2 suggested that these two glycosylated proteins were related and this was confirmed by tryptic peptide mapping. G2 was shown to be derived from G1 in pulse chase experiments and a similar relationship between two higher mobility polypeptides synthesized in the presence of tunicamycin was observed. The G protein may have a precursor since G1 did not appear immediately following a pulse labelling. The precursor could not however be identified. An additional minor glycosylated polypeptide of 42K was found to be related to the G protein. The F1 protein appeared to be poorly glycosylated and a difference in mobility of the polypeptide synthesized in the presence of tunicamycin did not appear to be directly due to a lack of N-linked oligosaccharides. The polypeptide migrated more slowly under non-reducing conditions but no evidence of a small disulphide bonded polypeptide was found in contrast to the situation with other paramyxoviruses. This polypeptide appeared to be the major PVM protein expressed on the cell surface and was associated with G1 and G2 as the major protein in a particulate fraction of the infected cell supernatant. Tentative relationships were suggested between the 39K, 35K and 25K polypeptides, the M and 24K polypeptides and the 20K and 19K polypeptides. This together with the observation that the 12K polypeptide was not a primary gene product suggested that there may be about 11 PVM polypeptides. The N or 39K and the 20K or 19K polypeptides were observed to be phosphorylated. Twelve possible TRT virus specific polypeptides of 150K, 129K, 95K, 83K, 57K, 45K, 38K, 35K, 3DK, 23K, 19K and 15K were identified. The 150K, 95K, 83K, 57K, 45K and 15K polypeptides were glycosylated with the latter three polypeptides showing a similar relationship to the F1,2, F1 and F2 polypeptides of paramyxoviruses. A broad glycosylated band designated the 31K polypeptide was identified that was similar to a smeared band observed on prolonged exposure of immunoprecipitates of PVM polypeptides labelled with [3H]-glucosamine. The 35K and 19K polypeptides were observed to be phosphorylated. PVM may be more closely related to RS virus than TRT virus since anti-PVM serum irnmunoprecipitated the RS virus N polypeptide but not any TRT virus polypeptides. The PVM 39K polypeptide and the RS virus P protein were recognised by a monoclonal antibody providing further evidence of a relationship between PVM and RS virus.

碩士<br>國立中興大學<br>獸醫學系<br>89<br>Summary Both swollen head syndrome and turkey rhinotracheitis are the respiratory diseases of poultry and caused by the avian pneumovirus. In Taiwan, the swollen head syndrome virus (SHSV) was first isolated from a chicken herd in 1991. The purpose of this study is to find the evolution trait of the Taiwan isolate as well as the virus distribution in the infected chickens. Avian pneumovirus consists of four subtypes, A, B, C and D. After amplification and sequencing of the F, G, M, N and P genes of the Taiwan/91 isolate by RT-PCR. The sequences were compared to other isolates in GenBank. Taiwanese isolate shared 99.1 to 99.8% similarities with the Hungary/89 and the Hungary/97 isolates of the subtype B. Sequence comparison of the most variable G gene region, indicated that the Taiwan isolate shared 99.1% similarity with the British isolate, but only shared 46.8% similarity with the French/85 isolate of the subtype D. Therefore, the Taiwan isolate of SHSV would be classified into the subtype B. The distinction diagnosis of the SHSV in clinic was executed by RT-PCR, whose primers designed of high conserved region of N, M, P. Meanwhile, the distinction diagnosis of different subtype of SHSV was token place with primers of F and G region. SPF chickens without APV antibody were inoculated with Taiwan isolate of SHSV, and their organs were collected from the 8th hours to the 42th days after inoculation for detection of the virus genome. The result showed that SHSV persisted for a long time in the infected chickens. Viral genome was first detected from the respiratory organs at the 8th hours post inoculation. All the other organs appeared positive, such as the sinus, brain, larynx, lung and lymphoid at the 7 — 9th days after inoculation. The positive organs were limited to thymus, Fabricius bursa, mesenteric lymph (GALT), caecum and tonsil at the 42th days post inoculation. Chicken sera were collected and tested with ELISA kit, indicating that the infected chickens turned to positive at the 10th days post infection. The serum neutralization antibodies appeared at the 9th days and the SN titer maintained 5 log2 at the 57th days post infection.

In dieser Arbeit wurde das PVM-Mausmodell verwendet, um die Bedeutung der Typ I und Typ III Interferonantwort für die Pathogenese einer pneumoviralen Infektion zu analysieren. Hierzu wurden zunächst mit Hilfe der reversen Genetik rekombinante PVM-Mutanten hergestellt, bei denen die Gene für die NS-Proteine, welche vermutlich als Interferonantagonisten fungieren, deletiert sind. Die Charakterisierung der Replikationsfähigkeit der rPVM dNS-Mutanten erfolgte in vitro in Interferon-kompetenten und Interferon-inkompetenten Zelllinien. Ein zentraler Schritt innerhalb dieser Charakterisierung war die Untersuchung der Induktion von Interferonen in vivo und in vitro nach Infektion mit den rPVM dNSMutanten, wobei nachgewiesen wurde, dass die NS-Proteine von PVM als Interferonantagonisten fungieren. In allen Interferon-kompetenten Zellkulturen wurde eine Attenuierung von rPVM dNS1, rPVM dNS2 und rPVM dNS1dNS2 bezogen auf rPVM beobachtet. In allen Interferon-inkompetenten Zellkulturen konnte die Attenuierung der rPVM dNS-Mutanten nahezu vollständig revertiert werden. Nach Infektion mit den rPVM dNS-Mutanten wurde in verschiedenen Zelllinien eine Induktion von Typ I und Typ III Interferonen betrachtet, wobei Unterschiede in der Stärke der Interferon-Induktion nach Infektion mit den rPVM dNS-Mutanten vorhanden waren. Zusammenfassend war es möglich, die NS1- und NS2-Proteine von PVM in Analogie zu RSV eindeutig als Antagonisten der Interferonantwort zu identifizieren. Die Untersuchung der protektiven Rolle von Typ I und Typ III Interferonen für die Replikation und Pathogenität von PVM bildete den zweiten Teil dieser Arbeit. Hierzu wurde die Replikationsfähigkeit und Pathogenität der rPVM dNS-Mutanten in verschiedenen Interferon-defizienten Mausstämmen getestet. Die Untersuchungen ergaben eine protektive Rolle von Typ I und Typ III Interferonen bei einer Infektion mit PVM, wobei den Typ I Interferonen ein effektiverer Einfluss zugeordnet werden konnte. Ein Vergleich von Replikation und Virulenz zwischen den verschiedenen Typ I oder Typ III oder Typ I/Typ III Interferonrezeptor-defizienten Mausstämmen belegte eine erhöhte Suszeptibilität der Typ I/Typ III Interferonrezeptor-defizienten Mäuse gegenüber einer Infektion mit den rPVM dNS-Mutanten. Eine vollständige Aufhebung der Attenuierung wurde auch in den Typ I/Typ III Interferonrezeptor-defizienten Mäusen nicht erlangt. Eine anti-apoptotische Funktion der NS-Proteine zusätzlich zu ihrer Wirkungsweise als Interferonantagonisten wurde aufgrund der unvollständigen Revertierung der Pathogenität der rPVM dNS-Mutanten in Typ I/Typ III Interferonrezeptor-defizienten Mäusen vermutet. Der abschließende Teil dieser Dissertation befasste sich mit der Frage, welche Zellen bei einer natürlichen pulmonalen Infektion Interferone in vivo produzieren. In vitro wurde beobachtet, dass überraschenderweise nur sehr wenige virusinfizierte oder uninfizierte Zellen Typ I Interferone bilden. Der Nachweis darüber, welche Zellen während einer pulmonalen Infektion hauptsächlich Interferone in vivo produzieren, war aufgrund der fehlenden Eignung der kommerziell erhältlichen Interferon-Antikörper für intrazelluläre Gewebefärbungen nicht möglich. Dennoch gelang es abschließend durch eine neue Nachweismethode erstmals Zellen mit rezeptorgebundenen Interferon zu identifizieren, wobei es sich um ziliierte Epithelzellen, Alveolarmakrophagen und vermutlich Clarazellen sowie Typ I und Typ II Pneumozyten handelte<br>In this thesis the PVM mouse model was used to unveil the role of type I and type III interferone response for the pathogenesis of pneumoviral infection. First, recombinant PVM dNS-mutants with single or combined deletions of the NS-genes were generated and replication efficiency characterization was analyzed in interferon-competent cells as well as in interferon-incompetent cells. A major point was to elucidate the NS-proteins of PVM as interferon antagonists. In interferon-competent cells the replication of rPVM dNS-mutants were significantly attenuated. These attenuation of all three rPVM dNS-mutants was almost completely reversed when interferon-incompetent cells were infected. Induction of type I and type III interferons was determined in different cell types after infection with rPVM dNS-mutants with differences in intensity of interferon induction. Thus, NS1- and NS2-proteins were clearly identified as interferon antagonists. To analyze the protective capacity of type I and type III interferons with respect to replication and pathology of PVM, mice lacking functional receptors for either or both interferons were infected with rPVM or the respective dNS-mutants. The results indicated that both type I and type III interferons which one restricted replication and pathology of PVM, with the former playing the greater role. Interestingly, the replication and virulence of wild-type PVM were completely unaffected by the presence or absence of functional receptors to type I and type III interferon, indicating that both systems are strongly suppressed during infection. However, pretreatment of mice with type I interferon were protective against lethal rPVM challenge, whereas pretreatment with type III interferon delayed but did not prevent death. Finally, the PVM-NS-proteins appeared to delay apoptosis independently of its IFN-antagonistic activity that may contribute to the limited pathogenicity of the viruses in type I/type III receptor deficient mice. In the last part type I interferon producing cells in a pneumoviral infection in vivo should be identified. It was documented that type I interferon producing cells are virusinfected cells as well as uninfected cells. Surprisingly, in vitro only few cells produced type I interferon during rPVM-GFP7 dNS1dNS2 infection. Because the commercially available interferon antibodies are not qualified for intracellular tissue staining it was not possible to identify in vivo the interferon producing cells, but by inventing a new method it was possible to verify the binding of type I interferon to the cell surface receptor of ciliated epithelial cells, alveolar macrophages and presumably Clara cells and type I and type II pneumocytes

The putative attachment protein G of pneumonia virus of mice (PVM), a member of the Pneumoviruses, is an important virulence factor with so far ambiguous function in a virus-cell as well as in virus-host context. The sequence of the corresponding G gene is characterized by significant heterogeneity between and even within strains, affecting the gene and possibly the protein structure. This accounts in particular for the PVM strain J3666 for which two differing G gene organizations have been described: a polymorphism in nucleotide 65 of the G gene results in the presence of an upstream open reading frame (uORF) that precedes the main ORF in frame (GJ366665A) or extension of the major G ORF for 18 codons (GJ366665U). Therefore, this study was designed to analyse the impact of the sequence variations in the respective G genes of PVM strains J3666 and the reference strain 15 on protein expression, replication and virulence. First, the controversy regarding the consensus sequence of PVM J3666 was resolved. The analysis of 45 distinct cloned fragments showed that the strain separated into two distinct virus populations defined by the sequence and structure of the G gene. This division was further supported by nucleotide polymorphisms in the neighbouring M and SH genes. Sequential passage of this mixed strain in the cell line standardly used for propagation of virus stocks resulted in selection for the GJ366665A-containing population in one of two experiments pointing towards a moderate replicative advantage. The replacement of the G gene of the recombinant PVM 15 with GJ366665A or GJ366665U, respectively, using a reverse genetic approach indicated that the presence of uORF within the GJ366665A significantly reduced the expression of the main G ORF on translational level while the potential extension of the ORF in GJ366665U increased G protein expression. In comparison, the effect of the G gene-structure on virus replication was inconsistent and dependent on cell line and type. While the presence of uORF correlated with a replication advantage in the standardly used BHK-21 cells and primary murine embryonic fibroblasts, replication in the murine macrophage cell line RAW 264.7 did not. In comparison, the GJ366665U variant was not associated with any effect on replication in cultured cells at all. Nonetheless, in-vivo analysis of the recombinant viruses associated the GJ366665U gene variant, and hence an increased G expression, with higher virulence whereas the GJ366665A gene, and therefore an impaired G expression, conferred an attenuated phenotype to the virus. To extend the study to other G gene organizations, a recombinant PVM expressing a G protein without the cytoplasmic domain and for comparison a G-deletion mutant, both known to be attenuated in vivo, were studied. Not noticed before, this structure of the G gene was associated with a 75% reduction in G protein expression and a significant attenuation of replication in macrophage-like cells. This attenuation was even more prominent for the virus lacking G. Taking into consideration the higher reduction in G protein levels compared to the GJ366665A variant indicates that a threshold amount of G is required for efficient replication in these cells. In conclusion, the results gathered indicated that the expression levels of the G protein were modulated by the sequence of the 5’ untranslated region of the gene. At the same time the G protein levels modulated the virulence of PVM<br>Das mutmaßliche „attachment“ Protein G des Pneumonievirus der Maus (PVM), einem Mitglied des Genus Pneumovirus, ist ein bedeutender Virulenzfaktor, mit allerdings noch nicht vollständig verstandener Funktion. Dabei zeichnet sich die Sequenz des G-Gens durch Nukleotid-Polymorphismen und damit verbundenen Variationen in der Genorganisation und möglicherweise der Proteinstruktur sowohl zwischen als auch innerhalb von PVM-Stämmen aus. Insbesondere für den PVM-Stamm J3666 wurden zwei verschiedene Organisationen des G-Gens beschrieben: ein Polymorphismus des Nukleotids 65 des G-Genes erzeugt einen neuen „upstream Open reading frame“ (uORF), der dem eigentlichen G-ORF vorausgeht (GJ366665A), oder führt zu einer Verlängerung des eigentlichen G-ORF von G um 18 Kodons (GJ366665U). Ziel dieser Studie war es deshalb, die Auswirkung dieser Sequenzvariabilitäten der für PVM J3666 beschriebenen G-Gene im Vergleich zu dem des Referenzstamms PVM 15 bezüglich Proteinexpression, der Virusreplikation und der Virulenz zu untersuchen. Als erstes wurden die beschriebenen Sequenzunterschiede bezüglich des PVM-Stamms J3666 untersucht. Die Analyse von 45 verschiedenen klonierten Fragmenten von PVM J3666 zeigte, dass es sich bei diesem Stamm eigentlich um zwei separate Viruspopulationen handelt, die sich durch die Sequenz und Struktur des G-Genes definieren lassen. Diese Unterscheidung wird durch weitere Nukleotid-Polymorphismen in den benachbarten Genen, M und SH, gestärkt. Sequenzielle Passagierung dieses gemischten Stammes in der standardmäßig zur Virusanzucht verwendeten BHK-21-Zelllinie resultierte in einem von zwei Experimenten in der Selektion der GJ366665A-Population, das ein Hinweis auf einen moderaten Replikationsvorteil darstellt. Der Austausch des G-Gens des Referenzstamms PVM 15 durch GJ366665A oder GJ366665U mithilfe der Reversen Genetik, zeigte, dass der uORF innerhalb von GJ366665A zu einer deutlich reduzierten Expression des eigentlichen G-ORF führt. Andererseits führte die potenzielle Verlängerung des ORF in GJ366665U zu einer im gleichen Maße erhöhten Expression des G-Proteins. Dagegen war der Einfluss der G-Genorganisation auf die Virusvermehrung in Zellkultur in Abhängigkeit von Zelllinie und Zelltyp inkonsistent. Während ein uORF mit einem Replikationsvorteil in BHK-21-Zellen und primären murinen embryonen Fibroblasten korrelierte, war dies in der murinen Makrophagen-Zelllinie RAW 264.7 nicht zu beobachten. Im Vergleich dazu konnte die GJ366665U-Variante nicht mit einem Einfluss auf die Virusvermehrung in Verbindung gebracht werden. Nichtsdestotrotz, konnte die GJ366665U-Variante, und damit eine erhöhte Expression von G, mit einer gesteigerten Virulenz assoziiert werden, während die GJ366665A-Variante, d. h. eine verringerte G-Expression zur Attenuierung des Virus führte. Die Untersuchungen wurden auf weitere G-Genstrukturen, d.h. ein rekombinantes PVM, rPVM-Gt, das ein N-terminal verkürztes G-Protein exprimiert, ausgeweitet. Zum Vergleich wurde eine Deletionsmutante des kompletten G-Gens, rPVM-ΔG, mit einbezogen. Von beiden Viren war bereits bekannt, dass sie in vivo attenuiert sind. Die Organisation des Gt-Gens war mit einer um 75 % verringerten Expression des entsprechenden Proteins assoziiert, was zuvor nicht beobachtet worden war. Zugleich zeigte rPVM-Gt eine deutliche Attenuierung der Replikation in RAW 264.7-Zellen und primären Mausmakrophagen, die von der G-Deletionsmutante noch übertroffen wurde. Die im Vergleich zu der GJ366665A-Variante deutlich höhere Reduktion der G-Expression dieser beiden G-Mutanten in Betracht ziehend, scheint dies darauf hinzuweisen, dass eine bestimmte Mindestexpression von G für eine effiziente Virusvermehrung in diesen Zellen benötigt wird. Zusammenfassend deuten die erhaltenen Ergebnisse darauf hin, dass die Expression des G-Proteins durch die jeweiligen 5’ nicht-translatierte Region des Gens moduliert wird, was einen neuen Mechanismus für Negativstrang-RNA-Viren darstellt. Zugleich moduliert die Expressionsrate von G die Virulenz von PVM