When I first arrived in Liège, the scientific activities of our laboratory focused on the identification of candidate-genes whose different alleles interfere with the resistance/susceptibility of animals against infectious diseases. Two genes were being intensively studied (Mx and OAS) due to their theoretical potential in interfering with the replicative cycle of several viruses responsible for bovines viral pneumonias (Baise et al., 2004 ; Gerardin et al., 2004 ; Leroy et al., 2005 ; 2006). My work consisted then, in the identification of other genes potentially implicated in the resistance against the Paramyxoviruses. The Paramyxoviridae family includes some of the great and ubiquitous disease-causing viruses of animals, including the bovine parainfluenza type 3, the bovine respiratory syncytial virus, the Newcastle disease virus, the distemper virus, etc. Evidence was accumulating that genetic factors were involved both in the control of infectious diseases (Abel et al., 1991 ; 1995 ; Alcais et al., 1997 ; Jin et al., 1999 ; Martinson et al. 1997 ; Shaw et al., 1995) and in the regulation of infection levels and clinical presentation (Garcia et al., 1999 ; Plancoulaine et al., 2000 ; 2003). Thus, identifying genes that control the organism response to paramyxoviruses was a crucial step in elucidating how they might affect the pathophysiological processes underlying the severity of the disease induced. Other experiences done in this laboratory showed us how risky and difficult was any extrapolation of the mouse results to another species if these results were brought through infection with a heterologous virus, so we decided to implement this strategy with Sendai virus (SeV), the archetype organism of the Paramyxoviridae family, from which most of the basic biochemical, molecular and biologic properties of the whole family were derived from (Chanock et al., 2001). With this goal in mind my thesis was divided in five successive steps: In order to establish a standard model of SeV infection, the first step consisted in determining the best volume of inoculum that was needed to achieve a safe, reproducible pulmonary deposition of Sendai virus in the mice lungs. Secondly we developed a murine model of SeV infection using a series of different and sophisticated procedures that allowed a quantitative assessment of disease severity and progression. Then we compared SeV infections among 6 strains of mice that were deliberately chosen because they originated from different lineages, as deduced from known genealogical and phylogenetic data. Applying these procedures to distinct inbred strains of mice, revealed highly significant differences in susceptibility between them. More specifically 129/Sv were highly susceptible while BALB/c were particularly resistant, BALB/c exhibiting a benign and asymptomatic affection of the epithelium of the airways, with no functional impact, generating slight mononucleated cell infiltration, in which viral replication is repressed and the virus swiftly eliminated. As a result, in the fourth part of my study we discussed a series of hypotheses that should be tested in the future to improve our understanding of why BALB/c is so resistant to SeV infection. Practically speaking, our studies led to the gathering of a genomic DNA collection from the parental extreme lines in terms of susceptibility (129/Sv) and resistance (BALB/c) and their F1 and F2 offspring. Within this bank, each of the 263 DNA sample is associated with a portfolio of phenotypic values that are estimators of the resistance each mouse opposed to the SeV. We hope that, between expert hands, this bank will allow the detection of genes of which the alleles contribute, at least in part, to the spectacular resistance of BALB/c. The last part of my thesis consisted in applying our model to establish if the receptor TLR4 influenced the pathophysiology of the Paramyxoviridae in general. Because the role of this receptor had already been excluded for SeV, we tested another virus of the same family and homologous for the mice, the PVM (standing for pneumonia virus of mice). This work showed, in contradiction of what had been found in heterologous models in the past, that TLR4 is not involved in host defense against respiratory tract infection with the Paramyxoviridae.

Apesar dos múltiplos avanços na compreensão gênica e taxonômica do Paramixovírus de serpentes (OPMV), apenas a patogenia pulmonar é razoavelmente conhecida nos viperídeos. O objetivo do presente estudo foi investigar, através de exames anátomo-patológicos, imunoistoquímicos, microbiológicos, hematológicos e sorológicos, a patogenia do Paramixovírus em jibóias (Boa constrictor). Dez animais foram inoculados por via endotraqueal com uma suspensão viral de OPMV. Os animais foram submetidos à eutanásia aos pares, aos 3, 7, 14, 21 e 60 dias após a infecção. Dois indivíduos foram utilizados como controle negativo. Lavados traqueais e amostras de sangue foram colhidas antes da inoculação, às necrópsias e nos animais dos grupos remanescentes. A presença de anticorpos anti-OPMV foi detectada aos 2 mPI através da técnica de inibição de hemaglutinação. A análise estatística dos resultados hematológicos demonstrou não haver diferença significativa entre os dados obtidos nos diversos tempos. À necrópsia amostras de órgãos foram colhidos para análises histopatológica, imunoistoquímica, bacteriológica e virológica (isolamento e RT-PCR). Macroscopicamente, apenas um animal (7dPI) apresentou pneumonia piogranulomatosa. As principais lesões microscópicas pulmonares observadas foram infiltração granulocítica, associada à formação de ninhos de células mononucleares, formação de sincícios; presença de hiperplasia e hipertrofia epiteliais em todos os tempos experimentais. Em pâncreas pôde ser diagnosticada formação de sincícios e presença de infiltrado mononuclear; em baço foi observada histiocitose, eventualmente associada à infiltração granulocítica perifolicular; gliose de padrão difuso ou focal. Os ensaios imunoistoquímicos e isolamento viral, com confirmação da presença do OPMV por RT-PCR, foram positivos em pulmão, fígado, baço e pâncreas dos 3 aos 21 dPI, sendo negativos aos 60 pPI. O diagnóstico molecular de lavado traqueal após passagem em cultura celular foram positivos aos 3, 7, 14 e 21d PI.. A ausência de sinais clínicos associada à detecção de lesões, isolamento e diagnóstico positivo por RT- PCR sugerem que as jibóias podem representar uma importante fonte assintomática de infecção até os 21 d PI. / Despite multiple advances in the genetic and taxonomic understanding of ophidian paramyxovirus (OPMV), only pulmonary pathogenesis is reasonably known in viperids. The objective of the present study was to investigate the pathogenesis of paramyxovirus infection in Boidae by pathological, imunohistochemical, microbiological, hematological and serological studies. Ten Boa constrictor snakes were infected by endotracheal inoculation with a viral solution. The animals were euthanatized in pairs at 3, 7, 14 and 21 days and at 2 months after infection. Two uninfected boas were sacrificed before and after the experimental study and were used as negative controls. Tracheal washes and blood were collected from all snakes. Seroconversion was detected at 2 mPI by hemagglutination inhibition assays. Estatistical analysis of the hematological data by Friedman Test revealed no diferences between them. At necropsy, samples of all major organs were obtained for histopathological, immunohistochemical, bacteriological and virological (viral isolation and RT-PCR). At necropsy, only one snake (7 days PI) had gross changes in the lung. The most consistent microscopic findings in the lungs were granulocyte infiltration, associated with the formation of mononuclear cell nests, formation of syncytia, and presence of epithelial hyperplasia and hypertrophy. Formation of syncytia was observed in pancreas, a mononuclear infiltrate was also observed; splenic histiocytosis with perifollicular granulocyte infiltration; diffuse and focal pattern of gliosis was detected in the CNS of most of the animals. Immunohistochemical examination and viral isolation, with confirmation of the virus\' presence by RT-PCR, were positive for lung, liver, spleen and pancreas from 3 to 21 dPI and negative at 2 m PI. Virus isolation from tracheal washes, with confirmation by molecular diagnosis were positive at times 3, 7, 14 and 21 dPI. At 2 mPI all results were negative. The immunohistochemical results associated with virus isolation and RT-PCR suggest that the virus was probably eliminated from the organism at 2 mPI. The absence of clinical symptoms associated with the detection of lesions and with isolation and a positive diagnosis by PCR in the present study suggest that Boa constrictors may represent an important source of infection for other reptiles.

Apesar dos múltiplos avanços na compreensão gênica e taxonômica do Paramixovírus de serpentes (OPMV), apenas a patogenia pulmonar é razoavelmente conhecida nos viperídeos. O objetivo do presente estudo foi investigar, através de exames anátomo-patológicos, imunoistoquímicos, microbiológicos, hematológicos e sorológicos, a patogenia do Paramixovírus em jibóias (Boa constrictor). Dez animais foram inoculados por via endotraqueal com uma suspensão viral de OPMV. Os animais foram submetidos à eutanásia aos pares, aos 3, 7, 14, 21 e 60 dias após a infecção. Dois indivíduos foram utilizados como controle negativo. Lavados traqueais e amostras de sangue foram colhidas antes da inoculação, às necrópsias e nos animais dos grupos remanescentes. A presença de anticorpos anti-OPMV foi detectada aos 2 mPI através da técnica de inibição de hemaglutinação. A análise estatística dos resultados hematológicos demonstrou não haver diferença significativa entre os dados obtidos nos diversos tempos. À necrópsia amostras de órgãos foram colhidos para análises histopatológica, imunoistoquímica, bacteriológica e virológica (isolamento e RT-PCR). Macroscopicamente, apenas um animal (7dPI) apresentou pneumonia piogranulomatosa. As principais lesões microscópicas pulmonares observadas foram infiltração granulocítica, associada à formação de ninhos de células mononucleares, formação de sincícios; presença de hiperplasia e hipertrofia epiteliais em todos os tempos experimentais. Em pâncreas pôde ser diagnosticada formação de sincícios e presença de infiltrado mononuclear; em baço foi observada histiocitose, eventualmente associada à infiltração granulocítica perifolicular; gliose de padrão difuso ou focal. Os ensaios imunoistoquímicos e isolamento viral, com confirmação da presença do OPMV por RT-PCR, foram positivos em pulmão, fígado, baço e pâncreas dos 3 aos 21 dPI, sendo negativos aos 60 pPI. O diagnóstico molecular de lavado traqueal após passagem em cultura celular foram positivos aos 3, 7, 14 e 21d PI.. A ausência de sinais clínicos associada à detecção de lesões, isolamento e diagnóstico positivo por RT- PCR sugerem que as jibóias podem representar uma importante fonte assintomática de infecção até os 21 d PI. / Despite multiple advances in the genetic and taxonomic understanding of ophidian paramyxovirus (OPMV), only pulmonary pathogenesis is reasonably known in viperids. The objective of the present study was to investigate the pathogenesis of paramyxovirus infection in Boidae by pathological, imunohistochemical, microbiological, hematological and serological studies. Ten Boa constrictor snakes were infected by endotracheal inoculation with a viral solution. The animals were euthanatized in pairs at 3, 7, 14 and 21 days and at 2 months after infection. Two uninfected boas were sacrificed before and after the experimental study and were used as negative controls. Tracheal washes and blood were collected from all snakes. Seroconversion was detected at 2 mPI by hemagglutination inhibition assays. Estatistical analysis of the hematological data by Friedman Test revealed no diferences between them. At necropsy, samples of all major organs were obtained for histopathological, immunohistochemical, bacteriological and virological (viral isolation and RT-PCR). At necropsy, only one snake (7 days PI) had gross changes in the lung. The most consistent microscopic findings in the lungs were granulocyte infiltration, associated with the formation of mononuclear cell nests, formation of syncytia, and presence of epithelial hyperplasia and hypertrophy. Formation of syncytia was observed in pancreas, a mononuclear infiltrate was also observed; splenic histiocytosis with perifollicular granulocyte infiltration; diffuse and focal pattern of gliosis was detected in the CNS of most of the animals. Immunohistochemical examination and viral isolation, with confirmation of the virus\' presence by RT-PCR, were positive for lung, liver, spleen and pancreas from 3 to 21 dPI and negative at 2 m PI. Virus isolation from tracheal washes, with confirmation by molecular diagnosis were positive at times 3, 7, 14 and 21 dPI. At 2 mPI all results were negative. The immunohistochemical results associated with virus isolation and RT-PCR suggest that the virus was probably eliminated from the organism at 2 mPI. The absence of clinical symptoms associated with the detection of lesions and with isolation and a positive diagnosis by PCR in the present study suggest that Boa constrictors may represent an important source of infection for other reptiles.

Human metapneumovirus (HMPV) is a worldwide respiratory pathogen that belongs to the paramyxovirus family of enveloped viruses and affects primarily the pediatric, geriatric, and immunocompromised populations. Despite its prevalence and importance to human health, no therapies are available against this pathogen. For paramyxoviruses, it is believed that infection starts by attachment of the virus to the surface of the cell through the viral attachment protein followed by fusion between the viral and cellular membranes, a process mediated by the fusion (F) protein at the plasma membrane and at neutral pH. Previous work showed that HMPV infection can occur in the absence of the attachment protein and membrane fusion triggered by the F protein can be promoted by low pH. The work presented here are significant advances in our understanding of the entry process of HMPV. We confirmed that the F protein has receptorbinding functions and identified the cellular binding partner to be heparan sulfate proteoglycans (HSPGs). Additionally, we provide evidence that electrostatic interactions at two different regions play important roles for the proper folding, stability, and low pH triggering of the HMPV F protein. We confirmed the hypothesis that protonation of H435 is important for HMPV F triggering and provide additional evidence that the entry of HMPV may be occurring through endocytosis. Therefore, we hypothesize that HMPV entry occurs through endocytosis after viral binding to HSPGs through the F protein and membrane fusion occurs in an acidified compartment.

Enveloped viruses, such as HIV, influenza, and Ebola, utilize surface glycoproteins to bind and fuse with a target cell membrane. This fusion event is necessary for release of viral genomic material so the virus can ultimately reproduce and spread. The recently emerged Hendra virus (HeV) is a negative-sense, single-stranded RNA paramyxovirus that presents a considerable threat to human health as there are currently no human vaccines or antivirals available. The HeV utilizes two surface glycoproteins, the fusion protein (F) and the attachment protein (G), to drive membrane fusion. Through this process, the F protein undergoes an irreversible conformational change, transitioning from a meta-stable pre-fusion conformation to a more thermodynamically stable post-fusion structure. Understanding the elements which control stability of the pre-fusion state and triggering to the post-fusion conformation is important for understanding F protein function. Studies that replace or mutate the TM domain of the F protein of several viruses implicated the TM domain in the fusion process, but the structural and molecular details in fusion remain unclear. Previously, analytical ultracentrifugation was used to demonstrate that isolated TM domains of HeV F protein associate in a monomer-trimer equilibrium. To determine factors driving this association, we analyzed the sequence of several paramyxovirus F protein TM domains and found a heptad repeat of β-branched residues. Analysis of the HeV F TM domain specifically revealed a heptad repeat leucine-isoleucine zipper motif (LIZ). Replacement of the LIZ with alanine resulted in dramatically reduced TM-TM association. Mutation of the LIZ in the whole protein resulted in decreased protein expression and pre-fusion conformation. To further understand the role of the TM domain, the TM domain was targeted as a potential modulator of F protein stability and function. Exogenous HeV F TM constructs were co-expressed with the full length F protein in Vero cells to analyze the effects on protein expression. Co-expression of the exogenous HeV F TM constructs dramatically reduced the expression of HeV F. However, the co-expression of exogenous HeV F TM constructs with a different paramyxovirus F protein, PIV5 F, did not strongly affect PIV5 F expression levels, suggesting that the interaction of the exogenous TM constructs is specific. Fusion assays revealed that HeV F TM constructs dramatically reduced HeV F, but not PIV5 F fusion activity. We hypothesize that the short exogenous HeV TM constructs associate with the TM domain from full-length HeV F, resulting in pre-mature triggering or protein misfolding. The work presented here demonstrates that specific elements in the TM domain contribute to TM association and pre-fusion protein stability. Furthermore, targeting these interactions may be a viable approach for antiviral development against this important pathogen.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Raymond R. R. Rowland / Emerging viral diseases threaten the health of the US swineherd and have the potential to impact the industry. Parvoviruses are capable of infecting birds, livestock and humans, however, in swine, parvoviruses cause reproductive failure and contribute to a devastating set of diseases termed porcine circovirus associated disease (PCVAD). Here, a divergent porcine parvovirus, porcine parvovirus 7 (PPV7), distantly related to known parvovirus sequences, was identified in market pigs in the US. The PPV7 non-structural protein displayed 42.4% similarity to Eidolon helvum parvovirus 2 and 37.9% similarity to turkey parvovirus. Conserved parvovirus replicase motifs including three rolling circle replication (RCR), two NTP-binding motifs and a helicase- binding domain, were present in PPV7. Analysis by qPCR of 182 porcine samples found 16 (8.6%) positive, suggesting moderate nucleic acid prevalence in US swine. Paramyxoviruses are capable of infecting various species including cattle, pigs and humans, causing respiratory disease and importantly, can overcome species barriers causing disease. In 2013, a novel paramyxovirus sequence was described in Hong Kong, China in slaughterhouse pigs, and subsequently named porcine parainfluenza virus 1 (PPIV1). The second study identifies two complete PPIV1 genomes in US pigs originating in Oklahoma and Nebraska that display 90.0-95.3% identity to the Chinese strains. Molecular analysis by qPCR resulted in 6.1% prevalence in 279 porcine respiratory samples. Further serological analysis revealed 66.1% of 59 porcine sera samples were positive by PPIV1 F ELISA. Eleven 3-week old nursery pigs from a PPIV1 naturally infected herd were monitored for signs of infection. No clinical signs were seen in the animals, however, six pigs and the lungs of one animal tested qPCR positive by the conclusion of the study. Taken together, PPIV1 is moderately prevalent in US swine-herds. Previously known to infect avian species, canines and swine, recent reports have identified circoviruses in bats, mink, and human feces. In pigs, porcine circovirus 2 (PCV2) is essential to PCVAD, a group of diseases including reproductive failure, respiratory disease complex (PRDC), porcine dermatitis and nephropathy syndrome (PDNS) and postweaning multisystemic wasting syndrome (PMWS). Additionally, PCV2 nucleic acid has been detected in mammalian species other than swine such as cattle and mink. The final study focuses on the identification and characterization of a divergent circovirus, porcine circovirus 3, identified in aborted mummies taken from sows displaying clinical and histological signs of PDNS. Putative capsid and replicase open reading frames display 37% and 55% identity to PCV2, respectively. A retrospective study of 48 PDNS cases, PCV2 negative by immunohistochemistry (IHC), identified 45 positive and 60% of a subset, positive for PCV3 by IHC. Molecular and serological prevalence studies revealed 12.5% nucleic acid and 55% antibody prevalence in US swine samples. Collectively, these studies identify emerging porcine viruses with the potential to cause disease using metagenomic sequencing. The results of these studies will help to mitigate the risk attributed to emerging swine viruses causing disease outbreaks.

Les paramyxovirus possèdent deux glycoprotéines d’enveloppe (gps): la protéine F, permettant la fusion avec la cellule hôte, et la protéine d’attachement appelée G, H ou HN. Les gps H et F d’une souche vaccinale du virus de la rougeole peuvent être incorporées sur des vecteurs lentiviraux (H/F-LV) permettant une transduction efficace des lymphocytes T et B humains non stimulés, habituellement réfractaires. Nous avons montré que les vecteurs H/F-LV sont capables de transduire des cellules B cancéreuses, activées et quiescentes, contrairement aux VSV-G-LV classiques. Leur utilisation in vivo est cependant confrontée à la présence d’anticorps neutralisants induits par la vaccination, dirigés majoritairement contre H. Après la mutation des 2 épitopes immunodominants de H, les vecteurs conservent leur tropisme et échappent à la neutralisation par les anticorps monoclonaux, mais sont toujours neutralisés par le sérum humain. Les souches émergentes de rougeole de génotype D, qui paraissent résister à la vaccination, présentent une glycosylation supplémentaire de la H. Introduite dans notre mutant, elle permet aux H/F-LV de transduire efficacement les cellules T et B en présence de sérum ou de sang total. Les pneumovirinae (le Virus Respiratoire Syncytial et le Métapneumovirus Humain (HMPV)) sont la première cause d’infections respiratoires chez le nourrisson, il n’existe pas de vaccin contre ces virus. Nous avons mis au point un système de Virus-Like Particle rétrovirales incorporant les gps F et G de HMPV (HMPV-VLPs). Injectées à des souris, les HMPV-VLP induisent une forte réponse d’anticorps neutralisants. De plus, suite à une épreuve virale, les souris sont protégées de l’infection par hMPV. / Paramyxoviruses contain two envelope glycoproteins : the F protein allowing fusion with the host cell and an attachment protein, called G, H or HN. Lentiviral vectors pseudotyped with the Edmonston measles virus hemagglutinin and fusion glycoproteins (H/F-LVs) allowed for the first time efficient transduction of quiescent human T and B cells. We showed that H/F-LVs were also able to efficiently transduce quiescent and activated cancer B cells, in contrast to the classical VSV-G-LVs. However, a major obstacle in the use of H/F-LVs in vivo is that most of the human population is vaccinated against measles inducing a humoral immune response exclusively directed against H. LVs pseudotyped with H-glycoproteins mutated in the 2 major epitopes escaped inactivation by monoclonal antibodies but were still neutralized by human serum. Consequently, we took advantage of newly emerged MV-D genotypes that were less sensitive to MV vaccination due to a different glycosylation pattern. The mutation responsible was introduced into the mutated H/F-LVs allowing efficient transduction of quiescent lymphocytes in the presence of high concentration of MV antibody-positive human serum or total blood. Pneumovirinae (Respiratory Syncitial Virus and human metapnemovirus (HMPV)) are the leading cause of respiratory infections in infants and no vaccine is available against these viruses. We designed retroviral Virus-Like Particle incorporating HMPV F and G gps (HMPV-VLPs). HMPV-VLPs injected to mice induce a strong neutralizing antibody immune response in vivo. Furthermore, upon a viral challenge, HMPV-VLP vaccinated mice are protected against hMPV infection.

Avian paramyxoviruses (APMVs) consist of twelve distinct serotypes (APMV-1 to -12) isolated from a wide variety of domestic and wild birds. APMV-1/Newcastle disease virus (NDV) is the most characterized and globally important avian pathogen, because of the huge economic loss associated with the disease. However, very little information is known about the pathogenicity of APMV 2-12. APMV expresses six structural and two accessory proteins. The functions of APMV accessory proteins (V and W) are not fully established. Only the function of V protein in NDV is studied so far. V protein was found to be an IFN antgonist and a major virulent determinant of NDV. In this study, we tested for the potential functions of W protein in NDV and fuctions of V protein in other APMV serotypes. Vaccination failure is a major cause for NDV outbreak in developing and tropical countries, because of thermolabile nature of vaccine strains. Thermostable and thermolabile NDV strains exhibit difference in W protein length. In the first part of our study, we mutated the genome of a thermolabile NDV strain to express W protein of different lengths, rescued recombinant viruses by reverse genetics system and tested for thermostability. Our results showed that W protein does not confer thermostability to NDV. In the second part of study, we constructed plasmids expressing APMV -2, -3 and -6V proteins and tested for IFN antagonism by a dual luciferase reporter assay. Our results showed that APMV-3V acts as IFN antagonist by blocking IFN induction and thereby may play an important role in the evasion of innate immunity. / Master of Science

Members of the family Paramyxoviridae are non-segmented, negative-strand RNA viruses. A large and diverse host species are infected by paramyxoviruses, including avian, porcine, canine, bovine, equine, ovine, reptiles, aquatic species and humans. In the last few decades, many novel paramyxoviruses have emerged causing catastrophic illnesses in different aquatic and terrestrial species of animals and some of them also made the species jump to humans. Two novel paramyxoviruses 81-19252 (Texas81) and 92-7783 (ISU92) were isolated in the 1980s and 1990s from the brain of pigs that experienced respiratory and central nervous system disease from South and North Central United States. To understand their importance as swine pathogens, molecular characterization and pathogenicity studies were undertaken. The complete genome of Texas81 virus was 15456 nucleotides (nt) and ISU92 was 15480 nt in length consisting of six non-overlapping genes coding for the nucloeo- (N), phospho- (P), matrix (M, fusion (F), hemagglutinin-neuraminidase (HN) and large polymerase (L) proteins in the order 3'-N-P/C/V-M-F-HN-L-5'. The features related to virus replication and found to be conserved in most members of Paramyxoviridae were also found in swine viruses. These include: conserved and complementary 3â leader and 5â trailer regions, trinucleotide intergenic sequences, highly conserved gene start and gene stop signal sequences. The length of each gene of these two viruses was similar except for the F gene, in which ISU92 had an additional 24 nt "U" rich 3â untranslated region (UTR). The P gene of these viruses were predicted to express the P protein from the primary transcript and edit a portion of its mRNA to encode V and D proteins and the C protein was expected to be expressed from alternate translation initiation from the P gene as in Respiroviruses. Sequence specific features related to virus replication and host specific amino acid signatures in P, F, HN and L proteins indicated that these viruses probably originated from bovine parainfluenzavirus 3. Pairwise comparisons of deduced amino acid sequences of swine viral proteins with members of Paramyxoviridae and phylogenetic analysis based on individual genes as well as predicted amino acid sequences suggested that these viruses were novel members of the genus Respirovirus of the Paramyxovirinae subfamily and genotype A of bovine parainfluenzavirus type 3. The mild clinical signs and undetectable gross and microscopic lesions observed in swine parainfluenzavirus (sPIV3)-infected pigs indicate the inapparent nature of these viruses in pigs. Limited seroprevalence studies in serum samples collected from pig farms in Minnesota and Iowa in 2007-2008 by indirect ELISA revealed that sPIV3 are not circulating in these farms. The mild pathogenicity of sPIV3 can facilitate its development as a vaccine vector. The screening ELISA developed by us could be used to detect seroprevalence of sPIV3 in animal and human populations. / Master of Science

Made available in DSpace on 2014-08-20T14:38:03Z (GMT). No. of bitstreams: 1 dissertacao_cristina_nunes.pdf: 1093744 bytes, checksum: 0c42a534dae5175850fd939832f086a8 (MD5) Previous issue date: 2011-02-28 / Currently, the drug industry looks for new drugs based on natural products, for the production of drugs more efficient, for which the microorganisms did not show resistance to both humans and animals. A natural product that has been the subject of intense pharmacological and chemical studies by scientists for the control of diseases is propolis, a resinous substance produced by honeybees from exudates collected from different parts of the plant, which has been used for centuries in popular medicine due to its therapeutic properties. Chemical studies revealed the complex chemical composition, identifying in some cases more than 300 components including various bioactive phenolic compounds responsible for the virucidal action. This work initially describes the standardization of an ethanol extract of green propolis (EEPV), where the chemicals were identified by high performance liquid chromatography (HPLC), phytochemical characterization by thin layer chromatography (TLC), soluble solids, content of phenolics and flavonoids and antioxidant activity by 2.2 diphenil picryl hydrazyl (DPPH). The EEPV was also evaluated in vitro and in vivo for their capacity lentogenic virucidal against a strain of the virus of Newcastle disease (NDV) at two different temperatures (22 and 37 ° C), 5 incubation periods (0, 1, 2, 4 and 8 hours) of NDV in five different concentrations of EEPV (4000μg/dose, 400μg/dose, 40μg/dose, and 4μg/dose 0μg/dose). The EEPV standard is within the standards required by the MAP, with high levels of phenolics and flavonoids (12.93 and 6.05% respectively) as shown by HPLC, which identified high concentrations of phenolic acids (p-coumaric acid, hydroxycinnamic acid diprenyl , cinâmino acid derivatives), which are assigned the antibacterial, antioxidant, antiviral and virucidal. This extract showed dose-dependent virucidal activity (4000μg/dose e 400μg/dose) and time of incubation with the virus (2 hour). The inhibitory activity of EEPV against the strain of NDV lentogenic found in the present study suggests the use of this extract as an alternative to fight the infection by this virus. / Atualmente, a indústria farmacêutica busca novos medicamentos com base em produtos naturais, visando à produção de fármacos mais eficientes, para os quais os microrganismos não apresentem resistência, tanto para humanos quanto para animais. Um dos produtos naturais que tem sido objeto de intensos estudos farmacológicos e químicos por cientistas para o controle de enfermidades é a própolis, uma substância resinosa produzida por abelhas melíferas a partir de exsudatos coletados em diferentes partes das plantas, que tem sido utilizada durante séculos na medicina popular devido as suas propriedades terapêuticas. Estudos químicos revelaram a complexa composição da própolis, identificando em alguns casos mais de 300 componentes, incluindo vários compostos bioativos fenólicos responsáveis pela ação virucida. Este trabalho inicialmente descreve a padronização de um extrato etanólico de própolis verde (EEPV), onde foram identificados os compostos químicos por cromatografia liquida de alta eficiência (CLAE), caracterização fitoquímica por cromatografia em camada delgada (CCD), teor de sólidos solúveis, teor de fenóis e flavonóides totais e atividade antioxidante por 2,2 diphenil picril hidrazil(DPPH). O EEPV foi avaliado também in vitro e in vivo, quanto a sua capacidade virucida contra uma cepa lentogênica do vírus da doença de Newcastle (NDV) em duas temperaturas distintas (22 e 37°C), 5 períodos de incubação (0, 1, 2, 4 e 8 horas) do NDV em 5 concentrações de EEPV distintos (4000μg/dose, 400μg/dose, 40μg/dose, 4μg/dose e 0μg/dose). O EEPV padronizado está dentro dos padrões requisitados pelo MAPA, com altos níveis de fenóis e flavonóides totais (12.93 e 6,05% respectivamente), comprovado por CLAE, o qual identificou altas concentrações de ácidos fenólicos (ácido p-cumárico, ácido diprenil hidroxicinâmico, derivados do ácido cinâmino), os quais são atribuídos as propriedades antibacteriana, antioxidante, antiviral e virucida. Este extrato apresentou atividade virucida dependente da dose (4000μg/dose, 400μg/dose) e do tempo de incubação com o vírus (2 horas). A atividade inibitória do EEPV contra a cepa lentogênica de NDV, encontrada no presente estudo sugere a utilização deste extrato como uma alternativa no combate a infecções por este vírus.