Influência do herpesvirus bovino tipo 5 no semen congelado de bovino sobre os parâmetros espermáticos e desenvolvimento embrionário in vitro

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Detecção herpesvírus bovino tipo 5 em cortes histológicos e fragmentos de encéfalo congelado pela reação em cadeia de polimerase

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

A implicação do papillomavírus humano no câncer de laringe: revisão sistemática

Contexto: O câncer de laringe é um dos mais comuns em homens após os 50 anos, atinge mais a região da cabeça e pescoço, representando 25% dos tumores malignos que acometem esta área e 2% de todas as doenças malignas. Aproximadamente 2/3 desses tumores surgem na corda vocal verdadeira e 1/3 localiza-se acima das cordas vocais. A relação entre o HPV e as doenças das vias aéreas superiores tem sido conhecida por quase um século, mas apenas nas últimas três décadas têm a sua atividade como potencial oncogénico reconhecido na literatura. Tipos de HPV similares aos encontrados no colo uterino foram também observados no câncer de laringe, língua e orofaringe. Objetivos: Avaliar a frequência de HPV em amostras de câncer de laringe; identificar os genótipos de HPV presentes em amostra de câncer de laringe; estabelecer a relação entre o câncer de laringe e o HPV, como fator de risco. Métodos: Revisão sistemática de ensaios clínicos na qual descritores e sinônimos para Neoplasias Laríngeas e Infecções por Papillomavirus foram usados nas seguintes bases de dados eletrônicas, até Março de 2012: CENTRAL; MEDLINE (PUBMED); LILACS e SciELO. Três revisores selecionaram, avaliaram a qualidade metodológica e extraíram os dados de estudos considerados relevantes. Resultados: Estimativas individuais combinadas em uma metanálise, resultaram em diferença estatisticamente significativa de HPV entre casos, quando comparados aos controles, com maior probabilidade entre os casos (OR 4.26, IC a 95% de 2.05 a 8.87, P=0.004). A análise estatística sugere substancial heterogeneidade (I2) entre os estudos (I2>50%, P<0,1), que pode ser explicada pelo tipo de controle utilizado. O tipo viral mais frequente entre os casos foi o HPV 16 e entre os controles foram os tipos virais HPV-6 e o HPV-16 e -18. Conclusões: Os resultados desta meta-análise apoiam a hipótese do envolvimento do HPV no cancer de laringe, o que sugere que o HPV como fator de risco depende da diferenciação com os demais fatores e o método de identificação do DNA viral.

Padronização e aplicação da técnica de ELISA (Enzyme-linked immunosorbent assay) indireto com anticorpo de captura para a detecção de anticoepos contra o cicovírus suíno tipo 2

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Obtencao de animais negativos para Circovirus suino 2 oriundos de granjas positivas: estrategia de manejo

O objetivo do trabalho é descrever uma estratégia para a obtenção de animais negativos para o PCV2 oriundos de uma granja positiva para este vírus. Dezesseis leitões foram obtidos de fêmeas que tiveram os títulos de IgG anti-PCV2 e o DNA viral testados durante a gestação. Esses leitões, aos sete e dez dias de idade, foram transferidos para a unidade de pesquisa. Durante o período de 7 e 10 aos 49 e 52 dias de idade, amostras de soro, suabes nasal e fecal foram coletadas, a cada sete dias. Após esse período, três animais permaneceram na unidade de pesquisa e foram acompanhados dos 49 aos 114 dias de idade, com coletas realizadas a cada 28 dias. Não houve diferença significativa (p = 0,317) de viremia entre marrãs (n = 6) e porcas (n = 10). Com relação aos níveis de IgG, observou-se diferença significativa (p = 0,0213) entre porcas e marrãs. Os leitões (n = 16), obtidos de duas fêmeas, foram transferidos para a unidade de pesquisa. Os animais entre 7 e 10 dias e aos 49 e 52 dias de idade apresentaram queda de IgG e ausência de IgM anti-PCV2; e as amostras de soro, suabe nasal e fecal foram negativos para o DNA de PCV2. Após os 49 dias, nos três animais mantidos isolados, a detecção de IgG, IgM e DNA para PCV2 permaneceu negativa. Concluindo, a estratégia de manejo utilizada permitiu obter suínos negativos para PCV2 oriundo de granjas positivas para o agente.

Retroviroses em felinos domésticos

The retrovirus are recognized as pathogenic group of virus for domestic animals. The particularitities of these viruses are the necessity of the enzyme transcriptase reversa, for the conversion of the viral RNA in viral DNA (provirus) and the incorporation in the DNA of the cell, what it confers to the infection the lifetime character, due to all the infected cells present the provirus our DNA. Among the retroviruses in domestic felines, the leukaemia and immunossupressive virus represent the more important diseases. The main form of transmission of the virus of the FeLV is occur by close contact and the saliva presents high viral concentration. For the FIV, the main form of transmission is represented by wounds of bite. The retrovírus, replicate mainly in high metabolization cells. The infection for FeLV cause mieloproliferativas and degenerative illnesses, while the FIV are related imunossupressora illness. The treatment for these retroviroses is symptomatic associated to imunomodulatory drugs, none of these drugs are capable to eliminate the virus. For the prevention of these retrovirus are used vaccines. However only the vaccine against FeLV have showed efficiency. Thus , the more important measures in control of these diseases is prevent the contact between infected and health felines. The ain of present study was reviewed the more important aspects of retroviruses in domestic felines, with emphasis to virulence properties, epidemiology, fisiopathogeny, clinical manifestations, methods of diagnosis, therapy, and control measures

Progressive multifocal leukoencephalopathy in a patient without apparent immunosuppression.

An 80-year-old man with no history of an immune-compromising disorder was diagnosed with progressive multifocal leukoencephalopathy (PML). He presented with dysphagia and left-sided weakness; magnetic resonance imaging demonstrated marked signal abnormality in the subcortical white matter of the left frontal lobe and in the posterior limb of the right internal capsule. Polymerase chain reaction (PCR) analysis of the cerebrospinal fluid (CSF) was negative for John Cunningham (JC) virus. On brain biopsy, foamy macrophages infiltrating the white matter were identified, staining positive for anti-simian virus 40 antibodies. Postoperatively, PCR for JC viral DNA in the CSF was positive, establishing the diagnosis of PML. Extensive investigation for an occult immunocompromising disorder was negative. The patient's neurologic deficits rapidly increased throughout his hospital stay, and he died 3.5 months after his diagnosis.

Identification and characterization of the herpes simplex virus type 2 gene encoding the essential capsid protein ICP32/VP19c.

We describe the characterization of the herpes simplex virus type 2 (HSV-2) gene encoding infected cell protein 32 (ICP32) and virion protein 19c (VP19c). We also demonstrate that the HSV-1 UL38/ORF.553 open reading frame (ORF), which has been shown to specify a viral protein essential for capsid formation (B. Pertuiset, M. Boccara, J. Cebrian, N. Berthelot, S. Chousterman, F. Puvian-Dutilleul, J. Sisman, and P. Sheldrick, J. Virol. 63: 2169-2179, 1989), must encode the cognate HSV type 1 (HSV-1) ICP32/VP19c protein. The region of the HSV-2 genome deduced to contain the gene specifying ICP32/VP19c was isolated and subcloned, and the nucleotide sequence of 2,158 base pairs of HSV-2 DNA mapping immediately upstream of the gene encoding the large subunit of the viral ribonucleotide reductase was determined. This region of the HSV-2 genome contains a large ORF capable of encoding two related 50,538- and 49,472-molecular-weight polypeptides. Direct evidence that this ORF encodes HSV-2 ICP32/VP19c was provided by immunoblotting experiments that utilized antisera directed against synthetic oligopeptides corresponding to internal portions of the predicted polypeptides encoded by the HSV-2 ORF or antisera directed against a TrpE/HSV-2 ORF fusion protein. The type-common immunoreactivity of the two antisera and comparison of the primary amino acid sequences of the predicted products of the HSV-2 ORF and the equivalent genomic region of HSV-1 provided evidence that the HSV-1 UL38 ORF encodes the HSV-1 ICP32/VP19c. Analysis of the expression of the HSV-1 and HSV-2 ICP32/VP19c cognate proteins indicated that there may be differences in their modes of synthesis. Comparison of the predicted structure of the HSV-2 ICP32/VP19c protein with the structures of related proteins encoded by other herpes viruses suggested that the internal capsid architecture of the herpes family of viruses varies substantially.

Regulatory function of in vivo anergized CD4+ T cells

It has been suggested that anergic T cells may not be only inert cells but may rather play an active role, for example by regulating immune responses. We have previously reported the existence of “anergic” IL-10-producing CD4+ T cells generated in vivo by continuous antigenic stimulation. Using a gene transfer system where the antigen recognized by such T cells is expressed in skeletal muscle by two different DNA viral vectors, we show that these cells not only remain tolerant toward their cognate antigen but also can suppress the immune response of naïve T cells against the immunogenic adenoviral proteins. Furthermore, they can completely inhibit tissue destruction that takes place as a result of an immune response. The system presented here is unique in that the T cells have been anergized in vivo, their antigen specificity and functional status are known, and the amount, form, and timing of antigen expression can be manipulated. This model will therefore permit us to carefully dissect the mechanisms by which these anergic T cells regulate the priming and/or effector function of naïve T cells.

Zalpha-domains: At the intersection between RNA editing and innate immunity

The involvement of A to I RNA editing in antiviral responses was first indicated by the observation of genomic hyper-mutation for several RNA viruses in the course of persistent infections. However, in only a few cases an antiviral role was ever demonstrated and surprisingly, it turns out that ADARs - the RNA editing enzymes - may have a prominent pro-viral role through the modulation/down-regulation of the interferon response. A key role in this regulatory function of RNA editing is played by ADAR1, an interferon inducible RNA editing enzyme. A distinguishing feature of ADAR1, when compared with other ADARs, is the presence of a Z-DNA binding domain, Zalpha. Since the initial discovery of the specific and high affinity binding of Zalpha to CpG repeats in a left-handed helical conformation, other proteins, all related to the interferon response pathway, were shown to have similar domains throughout the vertebrate lineage. What is the biological function of this domain family remains unclear but a significant body of work provides pieces of a puzzle that points to an important role of Zalpha domains in the recognition of foreign nucleic acids in the cytoplasm by the innate immune system. Here we will provide an overview of our knowledge on ADAR1 function in interferon response with emphasis on Zalpha domains.

The capsid protein of beak and feather disease virus binds to the viral DNA and is responsible for transporting the replication-associated protein into the nucleus

Circoviruses lack an autonomous DNA polymerase and are dependent on the replication machinery of the host cell for de novo DNA synthesis. Accordingly, the viral DNA needs to cross both the plasma membrane and the nuclear envelope before replication can occur. Here we report on the subcellular distribution of the beak and feather disease virus (BFDV) capsid protein (CP) and replication-associated protein (Rep) expressed via recombinant baculoviruses in an insect cell system and test the hypothesis that the CP is responsible for transporting the viral genome, as well as Rep, across the nuclear envelope. The intracellular localization of the BFDV CP was found to be directed by three partially overlapping bipartite nuclear localization signals (NLSs) situated between residues 16 and 56 at the N terminus of the protein. Moreover, a DNA binding region was also mapped to the N terminus of the protein and falls within the region containing the three putative NLSs. The ability of CP to bind DNA, coupled with the karyophilic nature of this protein, strongly suggests that it may be responsible for nuclear targeting of the viral genome. Interestingly, whereas Rep expressed on its own in insect cells is restricted to the cytoplasm, coexpression with CP alters the subcellular localization of Rep to the nucleus, strongly suggesting that an interaction with CP facilitates movement of Rep into the nucleus. Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Chemical studies of viral entry mechanisms: I. Hydrophobic protein-lipid interactions during sendai virus membrane fusion. II. Kinetics of bacteriophage λ DNA injection.

Viruses possess very specific methods of targeting and entering cells. These methods would be extremely useful if they could also be applied to drug delivery, but little is known about the molecular mechanisms of the viral entry process. In order to gain further insight into mechanisms of viral entry, chemical and spectroscopic studies in two systems were conducted, examining hydrophobic protein-lipid interactions during Sendai virus membrane fusion, and the kinetics of bacteriophage λ DNA injection.

Sendai virus glycoprotein interactions with target membranes during the early stages of fusion were examined using time-resolved hydrophobic photoaffinity labeling with the lipid-soluble carbene generator3-(trifluoromethyl)-3-(m-^(125 )I] iodophenyl)diazirine (TID). The probe was incorporated in target membranes prior to virus addition and photolysis. During Sendai virus fusion with liposomes composed of cardiolipin (CL) or phosphatidylserine (PS), the viral fusion (F) protein is preferentially labeled at early time points, supporting the hypothesis that hydrophobic interaction of the fusion peptide at the N-terminus of the F_1 subunit with the target membrane is an initiating event in fusion. Correlation of the hydrophobic interactions with independently monitored fusion kinetics further supports this conclusion. Separation of proteins after labeling shows that the F_1 subunit, containing the putative hydrophobic fusion sequence, is exclusively labeled, and that the F_2 subunit does not participate in fusion. Labeling shows temperature and pH dependence consistent with a need for protein conformational mobility and fusion at neutral pH. Higher amounts of labeling during fusion with CL vesicles than during virus-PS vesicle fusion reflects membrane packing regulation of peptide insertion into target membranes. Labeling of the viral hemagglutinin/neuraminidase (HN) at low pH indicates that HN-mediated fusion is triggered by hydrophobic interactions, after titration of acidic amino acids. HN labeling under nonfusogenic conditions reveals that viral binding may involve hydrophobic as well as electrostatic interactions. Controls for diffusional labeling exclude a major contribution from this source. Labeling during reconstituted Sendai virus envelope-liposome fusion shows that functional reconstitution involves protein retention of the ability to undergo hydrophobic interactions.

Examination of Sendai virus fusion with erythrocyte membranes indicates that hydrophobic interactions also trigger fusion between biological membranes, and that HN binding may involve hydrophobic interactions as well. Labeling of the erythrocyte membranes revealed close membrane association of spectrin, which may play a role in regulating membrane fusion. The data show that hydrophobic fusion protein interaction with both artificial and biological membranes is a triggering event in fusion. Correlation of these results with earlier studies of membrane hydration and fusion kinetics provides a more detailed view of the mechanism of fusion.

The kinetics of DNA injection by bacteriophage λ. into liposomes bearing reconstituted receptors were measured using fluorescence spectroscopy. LamB, the bacteriophage receptor, was extracted from bacteria and reconstituted into liposomes by detergent removal dialysis. The DNA binding fluorophore ethidium bromide was encapsulated in the liposomes during dialysis. Enhanced fluorescence of ethidium bromide upon binding to injected DNA was monitored, and showed that injection is a rapid, one-step process. The bimolecular rate law, determined by the method of initial rates, revealed that injection occurs several times faster than indicated by earlier studies employing indirect assays.

It is hoped that these studies will increase the understanding of the mechanisms of virus entry into cells, and to facilitate the development of virus-mimetic drug delivery strategies.

Contribution of the C-terminal tri-lysine regions of human immunodeficiency virus type 1 integrase for efficient reverse transcription and viral DNA nuclear import

Affiliation: Zhujun Ao, Éric Cohen & Xiaojian Yao : Département de microbiologie et immunologie, Faculté de Médecine, Université de Montréal

Mature reverse transcriptase (p66/p51) is responsible for low levels of viral DNA found in human immunodeficiency virus type 1 (HIV-1)

Reverse transcription of the HIV RNA genome is thought to occur in the host cell cytoplasm after viral adsorption. However, viral DNA has been isolated in cell-free virus particles. We have quantitated by polymerase chain reaction (PCR) amplification the amount of viral DNA in virions as compared to RNA. Virus produced by proviral DNA transfections of cos-7 cells or by chronically-infected H9 cells; neither of which express the cell surface CD4 receptor, contained at least 1000 times more viral RNA than DNA. In contrast, only 60 times more RNA than DNA was present in virus particles produced by transfection of Jurkat cells, which were CD4-positive and thus potentially susceptible to superinfection. Protease-defective virus, carrying only the precursor of reverse transcriptase (RT) p160gag-pol, contained virtually no detectable DNA. These results indicate that only mature RT (p66/p51) and not its precursor (p160gag-pol) is responsible for the presence of viral DNA in HIV.