The proteome of the infectious bronchitis virus Beau-R Virion

Infectious bronchitis is a highly contagious respiratory disease of poultry caused by the coronavirus IBV. It was thought that coronavirus virions were composed of three major viral structural proteins, until investigations of other coronaviruses showed that coronavirus virions also include viral non-structural and group specific proteins as well as host cell proteins. To study the proteome of IBV virions, virus was grown in embryonated chicken eggs and purified by sucrose gradient ultracentrifugation and analysed by mass spectrometry proteomic. Analysis of three preparations of purified IBV yielded the three expected structural proteins plus thirty-five additional virion-associated host proteins. Virion-associated host proteins had a diverse range of functional attributions, being involved in cytoskeleton formation, RNA binding and protein folding pathways. Some of these proteins were unique to this study, whilst others were found to be orthologous to proteins identified in SARS-CoV virions, and also virions from a number of other RNA and DNA viruses. Together these results demonstrate that coronaviruses have the capacity to incorporate a substantial variety of host protein, which may have implications for the disease process.

Syndromic approach to arboviral diagnostics for global travelers as a basis for infectious disease surveillance

Background Arboviruses have overlapping geographical distributions and can cause symptoms that coincide with more common infections. Therefore, arbovirus infections are often neglected by travel diagnostics. Here, we assessed the potential of syndrome-based approaches for diagnosis and surveillance of neglected arboviral diseases in returning travelers. Method To map the patients high at risk of missed clinical arboviral infections we compared the quantity of all arboviral diagnostic requests by physicians in the Netherlands, from 2009 through 2013, with a literature-based assessment of the travelers’ likely exposure to an arbovirus. Results 2153 patients, with travel and clinical history were evaluated. The diagnostic assay for dengue virus (DENV) was the most commonly requested (86%). Of travelers returning from Southeast Asia with symptoms compatible with chikungunya virus (CHIKV), only 55% were tested. For travelers in Europe, arbovirus diagnostics were rarely requested. Over all, diagnostics for most arboviruses were requested only on severe clinical presentation. Conclusion Travel destination and syndrome were used inconsistently for triage of diagnostics, likely resulting in vast under-diagnosis of arboviral infections of public health significance. This study shows the need for more awareness among physicians and standardization of syndromic diagnostic algorithms

Identification and characterization of antigenic proteins potentially expressed during the infectious process of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis causes paracoccidioidomycosis (PCM), a systemic mycosis presenting clinical manifestations ranging from mild to severe forms. A P. brasiliensis cDNA expression library was produced and screened with pooled sera from PCM patients adsorbed against antigens derived from in vitro-grown P. brasiliensis yeast cells. Sequencing DNA inserts from clones reactive with PCM patients sera indicated 35 open reading frames presenting homology to genes involved in metabolic pathways, transport, among other predicted functions. The complete cDNAs encoding aromatic-L-amino-acid decarboxylase (Pbddc), lumazine synthase (Pbls) and a homologue of the high affinity copper transporter (Pbctr3) were obtained. Recombinant proteins PbDDC and PbLS were obtained; a peptide was synthesized for PbCTR3. The proteins and the synthetic peptide were recognized by sera of patients with confirmed PCM and not by sera of healthy patients. Using the in vivo-induced antigen technology (IVIAT), we identified immunogenic proteins expressed at high levels during infection. Quantitative real time RTPCR demonstrated high transcript levels of Pbddc, Pbls and Pbctr3 in yeast cells infecting macrophages. Transcripts in yeast cells derived from spleen and liver of infected mice were also measured by qRT-PCR. Our results suggest a putative role for the immunogenic proteins in the infectious process of P. brasiliensis. (C) 2009 Elsevier Masson SAS. All rights reserved.

Risk factors for colonisation of newborn infants during an outbreak of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in an intermediate-risk neonatal unit

We describe a cross-sectional, survey to identify risk factors for colonisation of neonates by extended-spectrum P-Lactamase (ESBL)-producing Klebsiella pneumoniae. This occurred following exposure to a colonised healthcare worker during an outbreak in an intermediate-risk neonatal. unit. In total, 120 neonates admitted consecutively during a three-month period were screened for ESBL-producing K. pneumoniae by rectal swabbing and 27 were identified as colonised. Multivariate analysis showed colonisation to be independently associated with use of antibiotics and absence of breastfeeding. Previous use of antibiotics presented an odds ratio (OR) of 12.3 [95% confidence interval. (Cl): 3.66-41.2, P < 0.001]. The most commonly used antibiotics were penicillin and amikacin. Breastfeeding was associated with reduced risk for colonisation (OR: 0.22; 95% Cl: 0.05-0.99; P = 0.049). Nine isotates recovered during the first stage of the outbreak and 27 isolates from surveillance cultures were typed thereafter by pulsed-field gel electrophoresis, revealing six different profiles (A-F). Clones A, C, and E were implicated in the first stage of the outbreak, whereas among the 27 strains recovered from surveillance cultures, all six clones were identified. Clone A was also found on the hand of a nursing auxiliary with onychomycosis. We concluded that prior antimicrobial use predisposed to colonisation. The possible role of breastfeeding as a protective factor needs to be further elucidated. Detection of different genotypes of ESBL-producing K. pneumonioe suggests that dissemination of mobile genetic elements bearing the ESBL gene may have been superimposed on the simple dissemination of a clone during the outbreak. (c) 2008 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.

Plasmodium vivax: Microsatellite analysis of multiple-clone infections

We used mixtures of genomic DNA from two genetically distinct isolates from Brazil, 42M and 312M, to investigate how accurately 12-locus microsatellite typing describes the overall genetic diversity and characterizes multilocus haplotypes in multiple-clone Plasmodium vivax infections. We found varying PCR amplification efficiencies of microsatellite alleles; for example, from the same 1:1 mixture of 42M and 312M DNA we amplified predominantly 312M-type alleles at 10 loci and 42M-type alleles at 2 loci. All microsatellite alleles were accurately scored in 1:0.5 and 1:0.25 312M:42M DNA mixtures, even when minor peak heights did not meet previously suggested criteria for minor allele detection in multiple-clone infections. Relative proportions of major and minor alleles were unaffected by multiple displacement amplification of template DNA prior to PCR-based microsatellite typing. Although microsatellite typing may detect minor alleles in clone mixtures, amplification biases may lead to inaccurate assignment of predominant haplotypes in multiple-clone P. vivax infections. (C) 2008 Elsevier Inc. All rights reserved.

The Challenges of Antimicrobial Resistance in Brazil

Brazil is a country with continental proportions with high geographic and economic diversity. Despite its medical centers of excellence, antimicrobial resistance poses a major therapeutic challenge. Rates of methicillin-resistant Staphylococcus aureus are up to 60% and are related to an endemic Brazilian clone. Local resistance to vancomycin in Enterococci was first related to Enterococcus faecalis, which differs from European and American epidemiology. Also, local Klebsiella pneumoniae and Escherichia coli isolates producing extended-spectrum beta-lactamases have a much higher prevalence (40%-50% and 10%-18%, respectively). Carbapenem resistance among the enterobacteriaceae group is becoming a major problem, and K. pneumoniae carbapenemase isolates have been reported in different states. Among nonfermenters, carbapenem resistance is strongly related to SPM-1 (Pseudomonasaeruginosa) and OXA-23 (Acinetobacter baumannii complex) enzymes, and a colistin-only susceptible phenotype has also emerged in these isolates, which is worrisome. Local actions without loosing the global resistance perspective will demand multidisciplinary actions, new policies, and political engagement.

KClone: A Proposed Approach to Fast Precise Code Clone Detection

In all applications of clone detection it is important to have precise and efficient clone identification algorithms. This paper proposes and outlines a new algorithm, KClone for clone detection that incorporates a novel combination of lexical and local dependence analysis to achieve precision, while retaining speed. The paper also reports on the initial results of a case study using an implementation of KClone with which we have been experimenting. The results indi- cate the ability of KClone to find types-1,2, and 3 clones compared to token-based and PDG-based techniques. The paper also reports results of an initial empirical study of the performance of KClone compared to CCFinderX.

Nanotechnological delivery systems for the oral administration of active molecules: Polymeric microparticles and microemulsions applied to anti-inflammatory and anti-infectious drugs

This thesis was devoted to the development of innovative oral delivery systems for two different molecules. In the first part, microparticles (MPs) based on xylan and Eudragit® S- 100 were produced and used to encapsulate 5-aminosalicylic acid for colon delivery. Xylan was extracted from corn cobs and characterized in terms of its physicochemical, rheological and toxicological properties. The polymeric MPs were prepared by interfacial cross-linking polymerization and spray-drying and characterized for their morphology, mean size and distribution, thermal stability, crystallinity, entrapment efficiency and in vitro drug release. MPs with suitable physical characteristics and satisfactory yields were prepared by both methods, although the spray-dried systems showed higher thermal stability. In general, spraydried MPs would be preferable systems due to their thermal stability and absence of toxic agents used in their preparation. However, drug loading and release need to be optimized. In the second part of this thesis, oil-in-water microemulsions (O/W MEs) based on mediumchain triglycerides were formulated as drug carriers and solubility enhancers for amphotericin B (AmB). Phase diagrams were constructed using surfactant blends with hydrophiliclipophilic balance values between 9.7 and 14.4. The drug-free and drug-loaded MEs presented spherical non-aggregated droplets around 80 and 120 nm, respectively, and a low polydispersity index. The incorporation of AmB was high and depended on the volume fraction of the disperse phase. These MEs did not reduce the viability of J774.A1 macrophage-like cells for concentrations up to 25 μg/mL of AmB. Therefore, O/W MEs based on propylene glycol esters of caprylic acid may be considered as suitable delivery systems for AmB

Detection of infectious bronchitis virus and specific anti-viral antibodies using a concanavalin A-Sandwich-ELISA

Concanavalin A-Sandwich ELISA (Con A-S-ELISA) was developed for the detection of infectious bronchitis virus (IBV) or chicken specific anti-viral antibodies. The antigen detection limit for the Con A-S-ELISA was 10(5,1) EID50/mL. Three homologous and four heterologous IBV strains were similarly detected. This assay was highly effective in detecting the virus after infected tissue homogenates were passed once in embryonated chicken eggs, showing a good agreement with virus isolation technique. The Con A-S-ELISA was also used to measure anti-IBV chicken antibodies and showed a high coefficient of correlation (r = 0.85) and an agreement of k = 0.80 with the commercially available Indirect-ELISA. The relative sensitivity and specificity between these two tests were, respectively, 92.86% and 95.65% with an accuracy of 93.39%. Thus, the Con A-S-ELISA proved to be able to detect alternatively homologous and heterologous IBV strains or specific chicken anti-IBV antibodies, using the Con A as capture reagent of this assay.

Produtividade e qualidade dos frutos da laranjeira 'Pêra' clone IAC em 16 porta-enxertos na região de Bebedouro-SP

Avaliou-se a influência de 16 porta-enxertos na produtividade, nas características físicas e químicas (sólidos solúveis totais-°Brix; acidez; ratio; porcentagem de suco; índice tecnológico e tamanho dos frutos) dos frutos da laranjeira 'Pêra' [Citrus sinensis (L.) Osbeck] e na incidência e severidade da clorose variegada dos citros (CVC). O plantio do experimento foi realizado em julho de 1993, com espaçamento de 6,0 m entre linhas e 3,5 m entre plantas (476 plantas/ha). O experimento foi conduzido sem irrigação. O delineamento experimental foi em blocos ao acaso, duas plantas por parcela, três repetições e 16 tratamentos, constituídos pelas seguintes cultivares porta-enxertos: tangerineira 'Sun Chu Sha Kat' (Citrus reticulata Blanco), tangerineira 'Pectinífera' (C. reticulata), 'Shekwasha' (C. depressa Hayata), tangerineira 'Pectinífera/Shekwasha' (C. depressa Hayata), tangerineira 'Batangas' (C. reticulata), tangerineira 'Oneco' (C. reticulata), citrangor [citrange (Poncirus trifoliata Raf. x C. sinensis) x C. sinensis], citrandarin [C.sunki hort. Ex Tanaka) x Poncirus trifoliata (L.) Raf. cv. English, tangerineira 'Sunki' (C. sunki), tangerineira 'Suen-Kat' (C. sunki), tangerineira 'Nasnaran' (C. amblycarpa Ochse), tangerineira 'Venezuela' (C. reticulata), tangerineira Heen Naran (C. lycopersicaeformis hort. ex Tan. ), limoeiro 'Cravo' (C. limonia Osbeck) x tangerineira 'Cleópatra' (C. reshni hort ex Tanaka), limoeiro 'Cravo' (C. limonia), tangerineira 'Cleópatra' (C. reshni). A intensidade da clorose variegada dos citros variou em função dos porta-enxertos e não se relacionou com a produção de frutos até a quarta safra. Os porta-enxertos estudados, com exceção da tangerineira Nasnaran, proporcionaram qualidade e produções iniciais de frutos similares aos do limoeiro 'Cravo'.

Development and application of a Saccharomyces cerevisiae-expressed nucleocapsid protein-based enzyme-linked immunosorbent assay for detection of antibodies against infectious bronchitis virus

A Saccharomyces cerevisiae-expressed nucleocapsid (N) polypeptide of the M41 strain of infectious bronchitis virus (IBV) was used as antigen in a recombinant yeast-expressed N protein-based enzyme-linked immunosorbent assay (Y-N-ELISA). The Y-N-ELISA was rapid, sensitive, and specific for detecting chicken serum antibodies to IBV, and it compared favorably with a commercial ELISA.

Infectious bronchitis virus: detection and vaccine Strain differentiation by semi-nested RT-PCR

A semi-nested reverse transcription-polymerase chain reaction (Semi-N-RT-PCR) was developed and used to detect the S glycoprotein gene of infectious bronchitis virus (IBV) strains and to discriminate H120 vaccine strain from other strains. Viral RNA was extracted from the allantoic fluid of chicken embryos and from tissues of chickens experimentally infected with different strains of IBV. Amplification and identification of the viral RNA was performed using two sets of primers complementary to a region of the S glycoprotein gene in the Semi-N-RT-PCR assay. The pair of primers used in the first PCR consisted of universal oligonucleotides flanking a more variable region of S1-S2 gene. The second primer pair was used in the Semi-N-RT-PCR and was comprised of one of the primers from the first universal pair together with either another universal internal oligolucleotide or a oligonucleotide sequence specific for the H120 strain of IBV. The universal primers detected all reference IBV strains and field isolates tested herein. The Semi-N-RT-PCR had high sensitivity and specificity, and was able to differentiate the H120 vaccine strain from other reference IBV strains; including M41 strain. All tissue samples collected from chickens experimentally infected with H120 or M41 strains were positive in the semi-nested RT-PCR using universal primers, while only the H120-infected tissue samples were amplified by the set of primers containing the H120-oligonucleotide. In conclusion, the ability of Semi-N-RT-PCR to detect distinct IBV strains and preliminarily discriminate the vaccine strain (H120) closes a diagnostic gap and offers the opportunity to use comprehensive PCR procedures for the IBV diagnosis.

Effect of mannanoligosaccharides and/or enzymes on antibody titers against infectious bursal and Newcastle disease viruses

O efeito da inclusão de mananoligossacarídeo (MOS) e/ou enzimas em dietas de frangos sobre os títulos de anticorpos contra os vírus das doenças de Gumboro (VDG) e de Newcastle (VDN). Setecentos e cinqüenta aves foram distribuídas em um delineamento experimental inteiramente ao acaso, em arranjo fatorial 2 x 2 + 1, com dois níveis de MOS (0 e 0,1% até 21 dias e 0,05% de 22 até 42 dias de idade), dois níveis de enzimas (0 e 0,05%) e uma dieta-controle-positivo contendo antibióticos, totalizando cinco tratamentos com cinco repetições. Para análise dos anticorpos, amostras de sangue foram colhidas semanalmente por punção da veia jugular em duas aves de cada repetição. A primeira e a última colheita foram realizadas aos sete e 42 dias de idade, respectivamente. A inclusão de MOS resultou em aumento dos títulos contra VDG na quarta (P<0,03) e quinta (P<0,02) semanas, e contra VDN na terceira (P<0,01), quarta (P<0,03) e quinta (P<0,03) semanas de idade. O MOS foi efetivo em estimular a resposta imune humoral contra VDG e VDN vacinais.

A double antibody sandwich ELISA for rapid diagnosis of virus infection and to measure the humoral response against infectious bursal disease on clinical material

A double antibody sandwich ELISA (DAS-ELISA) was developed and employed for simultaneous direct detection of infectious bursal disease virus (IBDV) from bursal samples and to measure the humoral response, using the same basic immunoreagents, the purified and non-purified antigen, capture antibody and chicken hyperimmune sera were prepared, and standardized for this purpose, the DAS-ELISA was applied to both 80 bursal suspensions and 224 corresponding serum samples from vaccinated and non-vaccinated commercial hocks, Bursae samples were collected at 2 weeks of age, and submitted to histological examination, virus isolation in specific pathogen-free chickens embryos, and the DAS-ELISA technique, Serum titres obtained in indirect ELISA and serum neutralization test were compared with those in DAS-ELISA, the agreement was 80% between DAS-ELISA, and the conventional techniques, with high sensitivity (87%) and specificity (90%).