Introdução: As infecções respiratórias causam elevadas morbidade e mortalidade, sendo os vírus os principais agentes destas doenças. O monitoramento e vigilância de vírus respiratórios, desde os mais conhecidos até os emergentes, são importantes para a gestão em saúde, orientando tempo de profilaxia e minimizando o impacto de epidemias nas comunidades. Objetivos: Estudar a epidemiologia molecular do vírus sincicial respiratório (VSR) e descrever a epidemiologia dos seguintes vírus: influenza (IF), influenza A (H1N1), adenovírus (AdV) e parainfluenza (PIV) no Hospital de Clínicas de Porto Alegre. Para isso foram conduzidos três estudos: (1) caracterização das infecções respiratórias causadas por VSR, IF, AdV e PIV em crianças; (2) validação de uma técnica de reação em cadeia da polimerase em tempo real (RT-PCR) para detecção de VSR A/B e IF A/B e (3) caracterização de genótipos do VRS em crianças com infecções comunitárias e hospitalares. Métodos: No primeiro estudo foram levantadas as seguintes variáveis: casos de infecções respiratórias por VSR, AdV, PIV ou IF e H1N1; internações em enfermarias hospitalares e internações em unidades de terapia intensiva (UTI); infecções hospitalares e taxas de letalidade. As variáveis foram coletadas durante o atendimento de crianças (idade 0-12 anos) no HCPA entre 2007 e 2010. No segundo estudo, os alvos do ensaio de RT-PCR foram: o gene da proteína da matriz de IFA, o gene da hemaglutinina do IFB e o gene da nucleoproteína de RSVA/B. A especificidade do ensaio e seu limite de detecção foram determinados. Uma comparação entre RT-PCR e imunofluorescência indireta foi realizada. No terceiro estudo, 63 isolados de VSR (21 de origem nosocomial e 42 adquiridos na comunidade) foram sequenciados para estabelecer uma análise filogenética deste vírus. Resultados: Cada um dos vírus estudados apresentou um comportamento diferente. O VSR demonstrou ser o principal agente envolvido em infecções nosocomiais. Já os pacientes com AdV, bem como o VSR, apresentaram altas taxas de admissão em UTI em 2007 e 2010. O AdV e o H1N1 tiveram as maiores taxas de letalidade. O ensaio de RT-PCR mostrou-se específico e foi mais sensível do que a imunofluorescência, sendo capaz de detectar co-infecções. Os seguintes limites de detecção foram obtidos: 1 cópia/mL para a IFA, 10 cópias/mL para IFB, 5 cópias/mL para RSVA e 250 cópias/mL para RSVB. A investigação dos genótipos de VSR revelou que todos os isolados VSRA circulantes eram do mesmo grupo filogenético, o genótipo NA1 de origem japonesa. Por outro lado, os isolados VSRB foram distribuídos em grupos diferentes: BA4, POA1, POA2, POA3 e POA4. Este estudo foi o primeiro que descreveu a circulação do genótipo NA1 no Brasil, bem como quatro novos genótipos (POA1, POA2, POA3 e POA4). Conclusão: Os resultados obtidos no primeiro estudo demonstram o impacto das epidemias sazonais de vírus respiratórios. O segundo estudo corroborou relatos da literatura que técnicas moleculares, como RT-PCR, são adequadas para a detecção de vírus respiratórios. O terceiro estudo relatou genótipos emergentes de VSR. Estudos de vigilância como os descritos acima deveriam ser conduzidos periodicamente para acompanhar o padrão de comportamento dos vírus na população alvo. / Background: Respiratory tract infections of viral origin are a major cause of morbidity and mortality worldwide. Surveillance of well-known viruses and emerging threats is important for management, prophylaxis and to minimize impact on the community. Objective: To study the molecular epidemiology of respiratory syncytial virus (RSV) and describe the epidemiology of viruses: influenza (IF), influenza A (H1N1), adenovirus (AdV) and parainfluenza (PIV) at Hospital de Clinicas de Porto Alegre. Three studies were performed: (1) characterization of respiratory infections caused by RSV, IF, AdV and PIV in children, (2) validation of a technique of polymerase chain reaction in real-time (RT-PCR) to detect RSVA/B and IFA/B and (3) detection of genotypes of RSV in children with communityand hospital-acquired infection. Methods: In the first study, variables such as number of cases of viral (RSV, AdV, PIV or IF and H1N1) infection, hospitalizations in general wards and intensive care units (ICUs), nosocomial infections and lethality rates were collected. These variables obtained from each children (age 0-12 years) between 2007 and 2010. In the second study the assay RT-PCR was used to target the matrix gene of IFA, the hemagglutinin gene of IFB and the nucleoprotein gene of RSVA/B. The specificity of the assay and its limit of detection were determined. A comparison of RT-PCR and indirect immunofluorescence was performed. In the third study, 63 RSV isolates (21 nosocomial and 42 community-acquired) were submitted to sequencing to establish a phylogenetic analysis of this virus. Results: The different viruses presented a diversity of behaviors according to hospitalization, nosocomial outbreaks or lethality in children. RSV accounted for most nosocomial infections. Rates of ICU admission for AdV and RSV infection were highest in 2007 and 2010. During 2008–2009, H1N1 and AdV had the highest ICU admission rates. AdV and H1N1 had the highest lethality rates. The RT-PCR assay was more sensitive than immunofluorescence and it was able to detect viral co-infections. Futhermore, the limits of detection were 1 copy/μL for IFA, 10 copies/μL for IFB, 5 copies/μL for RSVA, and 250 copies/μL for RSVB. The genotyping study showed that hospital- and community-acquired RSVA isolates were from the same phylogenetic group (the same group of the NA1 Japanese isolates). Conversely, RSVB isolates were distributed across different groups: BA4, POA1, POA2, POA3 and POA4. This was the first study to describe circulation of the NA1 genotype in Brazil, as well as four RSVB genotypes: POA1, POA2, POA3 and POA4. Conclusion: The results obtained in the first study demonstrate the impact of seasonal epidemics of respiratory viruses. The second study confirmed that molecular techniques such as RT-PCR, are suitable for the detection of respiratory viruses. The third study reported emerging genotypes of RSV. Surveillance studies such as this should be performed periodically to monitor the behavior pattern of the virus in the target population.

As muitas formas diferentes de câncer representam uma grande dimensão no âmbito da saúde pública mundial. Embora os esforços da medicina diagnóstica, vários tumores permanecem sem resposta à terapia tradicional. Uma alternativa é o uso de terapia gênica, a qual consiste a transferência de um gene terapêutico para a célula tumoral com a expectativa de inibição da progressão tumoral. Nosso laboratório desenvolveu uma série de vetores adenovirais onde a expressão do transgene é controlada pela p53 e usamos esses vetores para mostrar que a presença de p19Arf (um parceiro funcional de p53) sensibiliza células de melanoma murino, B16-F10 (p53-tipo selvagem), associado à ação do interferão-beta (IFNbeta, uma citocina pleiotrópica) quando testado in vitro. Mesmo que os vetores adenovirais representem o sistema de transferência gênica mais utilizado para a terapia de genes de câncer, seu uso por via sistêmica é limitado principalmente por inativação pelo sistema imune. Diferentes técnicas visam proteger as partículas de vírus do sistema imunológico e direcioná-las para o leito tumoral. Uma dessas técnicas envolve a utilização de células estaminais mesenquimais (MSCs). As propriedades dos MSC incluem a auto renovação, o potencial de diferenciação, bem como a sua capacidade de migrar e infiltrar tumores. Para este fim, nosso objetivo era utilizar MSCs murinos como portadores de adenovírus que expressam IFNbeta e para verificar se a presença de p19Arf nas células tumorais aumentaria a sua sensibilidade para IFNbeta. Para itso, os CTMs foram isolados da medula óssea ou do tecido adiposo de ratinhos C57BL/ 6 machos. Foi verificada a presença de marcadores de CTM (Sca1, CD29) e a ausência de marcadores para linhagens hematopoiéticas (CD31, CD11b, CD45). Sendo as CTM do tecido adiposo foram mais fáceis de cultivar, estes foram utilizados nos seguintes ensaios. In vitro, a aplicação do vector adenoviral que codifica um gene repórter (eGFP) resultou em mais de 70% de eficiênciamde transdução de CTM, sem indução de alterações morfológicas até 72 horas após o tratamento. A aplicação de vector portador de IFNbeta também foi bem tolerada, no entanto transdução com p19Arf sozinho ou em combinação com IFNbeta induziu alterações morfológicas nas CTMs. Em seguida, as células B16-F10 foram transduzidas ou não com o vetor codificando p19Arf e co-cultivadas com MSCs que foram transduzidas ou não com IFNbeta, demonstrando que a presença de p19Arf confere sensibilidade aumentada de células B16-F10 ao tratamento com IFNbeta . Em ensaios preliminares, os tumores B16-F10 foram estabelecidos subcutaneamente em camundongos C57BL / 6 e, posteriormente, as MSC marcadas com eGFP foram aplicadas na circulação após a injeção da veia da cauda. Após 48 horas, estes tumores foram recuperados e a presença de células positivas para eGFP foi confirmada, indicando que os MSCs se infiltraram no microambiente do tumor / The many different forms of cancer represent a tremendous investment for public health all over the world. Although the efforts of both diagnostic and therapeutic medicine have reduced the number of deaths due to cancer, many tumor types remain impervious to traditional therapy. An alternative is the use of gene therapy which entails the transfer of a therapeutic gene to the tumor cells with the expectation of inhibiting tumor progression. Our laboratory has developed a series of adenoviral vectors where transgene expression is controlled by p53 and we have used these vectors to show that the presence of p19Arf (a functional partner of p53) sensitizes murine melanoma cells, B16-F10 (p53-wild type), to the action of interferon-beta (IFNbeta, a pleiotropic cytokine) when tested in vitro. Even though adenoviral vectors are the most utilized gene transfer system for cancer gene therapy, their systemic application is limited principally by immune inactivation. Different techniques aim to protect the virus particles from the immune system and to direct them to the tumor bed. One of these techniques involves the utilization of mesenchymal stem cells (MSCs). The properties of MSCs include self-renewal, the potential for differentiation as well as their ability to migrate to and infiltrate tumors. To this end, our objective was to utilize murine MSCs as carriers of adenovirus that express IFNbeta and to verify if the presence of p19Arf in the tumor cells would enhance their sensitivity to IFNbeta. For this, MSCs were isolated from bone marrow or adipose tissue from male C57BL/6 mice. The presence of MSC markers (Sca1, CD29) was verified as was the absence of markers for hematopoietic lineages (CD31, CD11b, CD45). Since the MSCs from adipose tissue were easier to cultivate, these were utilized in the following assays. In vitro, application of the adenoviral vector encoding a reporter gene (eGFP) at a multiplicity of infection of 1000 resulted in the transduction of more than 70% of the MSCs and without the induction of morphological alterations even by 72 hours post treatment. The application of a vector encoding IFN? was also well tolerated, however transduction with p19Arf alone or in combination with IFNbeta induced morphologic alterations in the MSCs. Next, B16-F10 cells were transduced or not with the vector encoding p19Arf and co-cultivated with MSCs that had been transduced or not with IFNbeta, demonstrating that the presence of p19Arf confers enhanced sensitivity of B16-F10 cells to the treatment with IFN?. In preliminary assays, B16-F10 tumors were established subcutaneously in C57BL/6 mice and later MSCs labeled with eGFP were applied in the circulation upon tail vein injection. After 48 hours, these tumors were recovered and the presence of eGFP-positive cells was confirmed, indicating that the MSCs infiltrated the tumor microenvironment

No description available.

PC12 rat pheochromocytoma cells respond to nerve growth factor (NGF) by neuronal differentiation and partial growth arrest. Mouse c-myc and adenovirus E1A genes were introduced into PC12 cells to study the influence of these nuclear oncogenes on neuronal differentiation. Expression of myc and E1A blocked morphological differentiation and caused NGF to stimulate rather than inhibit cell proliferation. NGF binding to cell surface receptors, activation of ribosomal S6 kinase, and ornithine decarboxylase induction were similar in myc and E1A expressing clones compared with wild-type PC12 cells, suggesting that changes in the cellular response to NGF were at a post-receptor level. The ability of myc and E1A expression to block the transcription-dependent induction of microtubule associated proteins by NGF further suggested that these genes may inhibit differentiation by interfering with NGP's ability to regulate transcription. These results illustrate that NGF can promote either growth or differentiation of PC12 cells, and that myc or E1A alter the phenotypic responses to growth factors.

<p>Vi&scaron;e od 100 vrsta virusa ljudi i životinja se izlučuje u spolja&scaron;nju sredinu. Prisustvo ovih virusa u povr&scaron;inskim vodama reflektuje fekalnu kontaminaciju i ukazuje na<br />opasnost za zdravlje ljudi i životinja. Na području Srbije se ne prati prisustvo patogenih virusa u povr&scaron;inskim vodama, pa&nbsp; čak ni u vodama za piće, a nije uspostavljena&nbsp; ni&nbsp; metodologija ovih ispitivanja. Shodno tome, cilj disertacije je da se utvrdi i analizira prisustvo animalnih i humanih virusa u povr&scaron;inskim vodama primenom najsavremenijih metoda koncentrovanja i detekcije virusa. U okviru disertacije ispitano je prisustvo sledećih virusa u povr&scaron;inskim vodama na teritoriji Vojvodine: humanih adenovirusa (HAdV); norovirusa (NoV) i hepatitis A virusa (HAV), adenovirusa svinja (PAdV),&nbsp; poliomavirusa goveda (BPyV) i hepatitis E virus (HEV).</p><p>Ispitano je ukupno 108 uzoraka povr&scaron;inskih i otpadnih voda koji su prikupljani od oktobra 2012. godine do juna 2014. godine. U radu su primenjene najsavremenije metode koncentrovanja i detekcije virusa u vodi, koje se u Srbiji nisu koristile za ovu namenu. Sprovedenim ispitivanjima dokazano je da su animalni i humani virusi prisutni u povr&scaron;inskim vodama na području Vojvodine. Najče&scaron;će detektovan&nbsp; virus u povr&scaron;inskim vodama je humani adenovirus (42,4%), a potom norovirusi GII i GI (40,4% i 15,2%), adenovirus svinja (11,1%), poliomavirus goveda (7,1%) i hepatitis E virus (3,0%). U ukupno 9 testiranih uzoraka gradske kanalizacione vode najče&scaron;će je detektovan HAdV (44,4%), NoV GII i GI&nbsp; (66,7% i 22,2%), BPyV je detektovan u samo jednom od 9 uzoraka, a niti u jednom nisu detektovani PAdV i HEV. Hepatitis A virus nije detektovan u uzorcima, a eksperimentalno je potvrđeno da su metode primenljive i za detekciju ovog virusa. Na osnovu rezultata prinosa procesne kontrole i utvrđenog prisustva virusa u uzorcima,&nbsp; zaključeno&nbsp; je da se ove metode mogu veoma uspe&scaron;no koristiti za detekciju virusne kontaminacije&nbsp; povr&scaron;inskih voda. Izvr&scaron;ena je igenotipizacija virusa iz odabranih uzoraka metodom sekvenciranja dela virusnog genoma. Indirektno je potvrđeno da su infekcije&nbsp;&nbsp; detektovanim virusima prisutne u populaciji životinja i ljudi. Prisustvo virusa u&nbsp;&nbsp; povr&scaron;inskim vodama i uzorcima gradske kanalizacije odražava infektivni status stanovni&scaron;tva, ali predstavlja i značajan rizik za zdravlje životinja i ljudi na području koje gravitira ispitanim vodama.&nbsp;</p> / <p>Over 100 types of pathogenic viruses are excreted in human and animal wastes. The presence of human and animal pathogenic enteric viruses in water environments reflects fecal contamination and indicates a risk to public health.&nbsp; Republic of Serbia does not implement surveillance for the presence of pathogenic human and animal viruses in surface waters and even in drinking water, neither is the established methodology of these studies in any institution in Serbia.&nbsp; Accordingly, the aim of the study was to determine and analyze the presence of human and animal viruses in surface water,&nbsp; using the latest methods&nbsp; of&nbsp; concentration and detection of the viruses.&nbsp; Within the dissertation examined the presence of the following viruses in surface waters in Vojvodina:&nbsp; Human adenoviruses&nbsp; (HAdV), noroviruses (NoV)&nbsp; and hepatitis A virus), Porcine adenovirus (PAdV) and Bovine polyomavirus (BPyV)&nbsp; and&nbsp; Hepatitis E virus (HEV).<br />A total of 108 samples of surface water and waste water were collected from October 2012 to June 2014. The paper are applied the most advanced methods and the concentration of virus detection in water, which in Serbia are not used for this purpose. The conducted tests have proven that the animal and human viruses present in surface waters in Vojvodina. The most commonly detected virus in surface water was human adenovirus (42.4%), followed by Norovirus GI&nbsp; and GII (40.4% and 15.2%),&nbsp; Porcine adenovirus&nbsp; (11,1%),&nbsp; Bovine polyomavirus&nbsp; (7.07%) and hepatitis E virus (3,0%).<br />In total of&nbsp; nine analysed sewage samples human adenovirus was detected in 44,4%&nbsp; of&nbsp; samples. The prevalence of norovirus GII and GI in sewage&nbsp; samples was&nbsp; 66,7%&nbsp; and 22,2%. Bovine&nbsp; polyomavirus was detected in one of nine samples while porcine adenovirus and hepatitis E virus were not detected in any of analyzed samples.&nbsp; Hepatitis A virus was not detected in samples, but&nbsp; it has been experimentally confirmed that the methods applicable for detection of the virus. Based on the results of process control and yield determined the presence of virus insamples, it was found that these methods can be successfully used to detect viral contamination of surface waters. Also, in these study was performed genotyping of viruses from selected samples by sequencing a part of the viral genome. Indirectly it is confirmed that the infection detected viruses present in a population of animals and humans. The presence of virus in samples of surface water and urban sewage reflects the infectious status of the population, but also constitutes a significant risk to the health of animals and people in the area that gravitates with tested waters.</p><p>&nbsp;</p>

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Submitted by Anderson Silva (avargas@icict.fiocruz.br) on 2012-12-27T16:54:47Z No. of bitstreams: 1 Tese completa Tatiana Prado doutorado BP pdf.pdf: 8757483 bytes, checksum: 8a4d7a0b6c6b6d917034f958c665cd1a (MD5) / Made available in DSpace on 2012-12-27T16:54:47Z (GMT). No. of bitstreams: 1 Tese completa Tatiana Prado doutorado BP pdf.pdf: 8757483 bytes, checksum: 8a4d7a0b6c6b6d917034f958c665cd1a (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / Este estudo teve como objetivo avaliar a presença de rotavírus (RVA), adenovírus (AdV), norovírus (NoV) e vírus da hepatite A (HAV) em amostras ambientais (esgoto e lodo de esgoto primário e tratado) geradas e obtidas em diferentes Estações de Tratamento de Esgoto (ETEs) da cidade do Rio de Janeiro pela detecção, quantificação e caracterização molecular, assim como determinar a eficiência de diferentes metodologias de recuperação viral em lodo de esgoto. A concentração destes vírus foi determinada em ETEs hospitalares que operam por processos anaeróbios (reator UASB e filtros anaeróbios) e aeróbio (lodo ativado de aeração prolongada) e em uma ETE urbana de grande porte (Alegria, Caju) que utiliza processo de lodo ativado convencional. Os resultados demonstraram que os efluentes hospitalares são contaminados com elevadas cargas virais, especialmente RVA, apontando para o risco da disseminação ambiental quando processos de tratamento biológicos convencionais (aeróbios e anaeróbios) não são adequados para a remoção viral. Uma grande variabilidade de genótipos virais foi encontrada nos efluentes hospitalares, particularmente para AdV, para os quais foram detectadas espécies C e D, comumente envolvidas em casos de infecções respiratórias e conjuntivite nosocomial. Apenas genótipo GII.4 de NoV foi detectado nos efluentes hospitalares, confirmando a prevalência desse genótipo no Brasil. No estudo que avaliou a detecção de HAV nas águas residuárias urbanas foi constatado que 58% das amostras de esgoto bruto foram positivas para HAV e as cargas de genomas virais não foram detectadas nos efluentes tratados do processo de lodo ativado convencional. A análise filogenética baseada na região do genoma VP1/2A de HAV detectados em 2009-2010 demonstrou a prevalência do subgenótipo IA e os isolados apresentaram maior identidade de nucleotídeos com isolados de HAV do Brasil e Argentina. A detecção viral obtida em diversas etapas da ETE de grande porte demonstrou a predominância de RVA e AdV e o sistema de lodo ativado convencional reduziu as cargas de genomas virais em 1 – 1,5 unidades log10 no efluente final tratado. A análise da eficiência dos métodos de detecção viral em lodo de esgoto demonstrou que a presença de inibidores nas amostras afeta o desempenho da amplificação de ácidos nucléicos e o uso do controle interno (bacteriófago PP7) pode ser adequado para monitorar as etapas de detecção viral. O método baseado na eluição utilizando extrato de carne foi o mais viável para recuperar os vírus pesquisados, sobretudo AdV em lodo de esgoto. Os AdV foram predominantes nas amostras de lodo de esgoto tratado e, portanto, poderiam ser considerados bons indicadores para a presença de vírus de disseminação entérica nesses resíduos. Esse é o primeiro estudo demonstrando a ocorrência de RVA e NoV GII em lodo de esgoto digerido anaerobicamente no Brasil, e espera-se que esse trabalho contribua para expandir essa linha de investigação, incluindo a análise de biossólidos produzidos por outros processos de estabilização. / The aim of this study was to evaluate the presence of rotavirus (RVA), adenovirus (AdV), norovirus (NoV) and hepatitis A virus (HAV) in environmental samples (sewage and primary and treated sewage sludge) produced in different wastewater treatment plants (WWTPs) in Rio de Janeiro city through the detection, quantification and molecular characterization, as well as, determining the efficiency of different viral recovery methods in sewage sludge. Viral concentrations were determined in hospital WWTPs that operates by anaerobic processes (UASB reactor and anaerobic filters) and aerobic (activated sludge process with prolonged aeration) and in a great urban WWTP (Alegria, Caju) that operates by conventional activated sludge process. The results demonstrate that hospital wastewaters and treated effluents by anaerobic and aerobic processes are contaminated with high viral loads, especially RV-A, pointing environmental dissemination risks when conventional biological treatment processes are not suitable for virus removal. A great variability of viral genotypes was found, mainly for AdV, which species C and D involved in respiratory infectious diseases and nosocomial keratoconjuctivites cases were detected. Only NoV GII.4 was obtained in hospital wastewaters, confirming the prevalence of this genotype in Brazil. In the study that evaluated HAV detection in urban wastewaters was demonstrated that 58% of the raw sewage samples were positive for HAV and the viral genome loads were not detected in the treated effluents from activated sludge process. Phylogenetic analysis based on VP1/2A genome region of HAV detected in 2009-2010 demonstrated the prevalence of subgenotype IA and the isolates showed higher nucleotide identity with HAV isolates from Brazil and Argentina. Virus detection determined in distinct stages of the conventional activated sludge process demonstrated the predominance of RVA and AdV and viral genome loads were reduced in 1 – 1,5 log10 units in the treated effluent. Analysis of the virus recovery methods efficiency in sewage sludge demonstrated that the presence of inhibitors affect the performance of the nucleic acid amplification reactions and the use of an internal control (PP7 bacteriophage) can be suitable to monitoring viral detection stages. The method based on beef extract elution was more viable for recovering viruses, mainly AdV in sewage sludge. Adenoviruses are predominant in these samples and, therefore, could be considered good indicators to evaluate the viral contamination in these residues. This is the first Brazilian report on the occurrence of RV-A and NoV GII in sewage sludge treated by anaerobic digestion and is expected that the present study contributes for the investigation of the enterically disseminated viruses in biosolids treated by other stabilization processes.

Human adenovirus (HAdV) is a common pathogen causing a broad spectrum of diseases. HAdV encodes the pVII protein, which is involved in nuclear delivery, protection and expression of viral DNA. To suppress the cellular interferon (IFN) and RNA interference (RNAi) systems, HAdVs encode non-coding virus-associated (VA) RNAs. In this thesis we have investigated the functional significance of the pVII protein and VA RNAI in HAdV-5 infected cells. We report that the propeptide module is the destabilizing element targeting the precursor pVII protein for proteasomal degradation. We also found that the Cul3-based E3 ubiquitin ligase complex alter the precursor pVII protein stability via binding to the propeptide sequence. In addition, we show that inhibition of the Cul3 protein reduces HAdV-5 E1A gene expression. Collectively, our results suggest a novel function for the pVII propeptide module and involvement of Cul3 in viral E1A gene expression. Our studies show that the cellular E3 ubiquitin ligase MKRN1 is a novel pVII interacting protein in HAdV-5 infected cells. MKRN1 expression reduced the pVII protein accumulation in virus-infected cells and affected infectious virus formation. Surprisingly, the endogenous MKRN1 protein underwent proteasomal degradation during the prolonged HAdV-5 infection. Furthermore, the precursor pVII protein enhanced MKRN1 self-ubiquitination, suggesting the direct involvement of pVII in the initiation of MKRN1 degradation. Hence, we propose that the MKRN1 is a novel antiviral protein and that HAdV-5 infection counteracts its antiviral activity. In papers III and IV, we tested the ability of various plant and animal virus encoded RNAi/miRNA and IFN suppressor proteins to functionally substitute for the HAdV-5 VA RNAI. Our results revealed that the Vaccinia virus E3L protein was able to partially substitute for the HAdV-5 VA RNAI functions in virus-infected cells. Interestingly, the E3L protein rescued the translational defect but did not stimulate viral capsid mRNA accumulation observed with VA RNA. Additionally, we show that the HAdV-4 and HAdV-37 VA RNAI are more effective in virus replication compared to HAdV-5 and HAdV-12 VA RNAI. In paper IV, we employed a novel triplex-specific probing assay, based on the intercalating and cleaving agent benzoquinoquinaxline 1,10-phenanthroline (BQQ-OP), to unravel triplex structure formation in 
VA RNAI. The BQQ-OP cleavage of HAdV-4 VA RNAI indicates that a potential 
triplex is formed involving the highly conserved stem 4 of the central domain and side 
stem 7. Further, the integrity of HAdV-4 VA RNAI stem 7 contributes to the virus growth in vivo.

Introduction: Epidemiological studies have indicated that 5-38% of influenza like illnesses (ILI) develop into severe disease due to, among others, factors such as; underlying chronic diseases, age, pregnancy, and viral mutations. There are suggestions that dual or multiple virus infections may affect disease severity. This study investigated the association between co-infection between influenza A viruses and other respiratory viruses and disease severity. Methodology: Datum for samples from North West England tested between January 2007 and June 2012 was analysed for patterns of co-infection between influenza A viruses and ten respiratory viruses. Risk of hospitalization to a general ward ICU or death in single versus mixed infections was assessed using multiple logistic regression models. Results: One or more viruses were identified in 37.8% (11,715/30,975) of samples, of which 10.4% (1,214) were mixed infections and 89.6% (10,501) were single infections. Among patients with influenza A(H1N1)pdm09, co-infections occurred in 4.7% (137⁄2,879) vs. 6.5% (59⁄902) in those with seasonal influenza A virus infection. In general, patients with mixed respiratory virus infections had a higher risk of admission to a general ward (OR: 1.43, 95% CI: 1.2 – 1.7, p = <0.0001) than those with a single infection. Co-infection between seasonal influenza A viruses and influenza B virus was associated with a significant increase in the risk of admission to ICU/ death (OR: 22.0, 95% CI: 2.21 – 219.8 p = 0.008). RSV/seasonal influenza A viruses co-infection also associated with increased risk but this was not statistically significant. For the pandemic influenza A(H1N1)pdm09 virus, RSV and AdV co-infection increased risk of hospitalization to a general ward, whereas Flu B increased risk of admission to ICU/ death, but none of these were statistically significant. Considering only single infections, RSV and hPIV1-3 increased risk of admission to a general ward (OR: 1.49, 95% CI: 1.28 – 1.73, p = <0.0001 and OR: 1.34, 95% CI: 1.003 – 1.8, p = 0.05) and admission to ICU/ death (OR: 1.5, 95% CI: 1.20 – 2.0, p = <0.0001 and OR: 1.60, 95% CI: 1.02 – 2.40, p = 0.04). Conclusion: Co-infection is a significant predictor of disease outcome; there is insufficient public health data on this subject as not all samples sent for investigation of respiratory virus infection are tested for all respiratory viruses. Integration of testing for respiratory viruses’ co-infections into routine clinical practice and R&D on integrated drugs and vaccines for influenza A&B, RSV, and AdV, and development of multi-target diagnostic tests is encouraged.

MSc (Microbiology) / Department of Microbiology / See the attached abstract below