L-asparaginase de Erwinia chrysanthemi: expressão proteica livre de células e bioconjugação a bacteriófagos / L-asparaginase from Erwinia chrysanthemi: cell-free protein expression and bioconjugation to bacteriophages

A L-Asparaginase (L-ASNase) de Erwinia chrysathemi (ErA) é uma enzima amplamente utilizada para o tratamento da leucemia linfoblástica aguda (LLA). Embora o seu uso como segunda linha de tratamento para a LLA tenha proporcionado consideráveis benefícios clínicos, reações de hipersensibilidade e rápida depuração plasmática ainda são problemas recorrentes. Ademais, extensivos e custosos processos de produção da ErA são necessários para a obtenção da enzima pura. Com base nesses problemas, o presente trabalho propõe (1) o estudo de viabilidade de expressão da ErA em um sistema de síntese proteica livre de células (SPLC) e (2) a conjugação da proteína em bacteriófagos como ferramenta alternativa para o isolamento e monitoramento da depuração plasmática da ErA. Foram utilizados extratos celulares de Escherichia coli suplementados com solução energética contendo creatina fosfato (CP) como fonte de energia para síntese in vitro de ErA. Para conjugação da ErA a bacteriófagos, o sistema SpyTag/SpyCatcher foi implementado: SpyCatcher foi fusionado à porção N-terminal da ErA e bacteriófagos filamentosos da linhagem M13 e fd foram modificados de modo a expressar SpyTag nas proteínas de capsídeo pIII e pVIII, respectivamente. Em relação ao primeiro objetivo, o sistema de SPLC foi capaz de expressar a ErA com atividade. A proteína foi expressa na fração solúvel e apresentou atividade enzimática significativamente superior em relação à reação controle (7,07 ± 0,68 U/mL vs. 1,83 ± 0,14 U/mL). Tempo necessário para obtenção do extrato celular foi reduzido de 45 para 26 hrs, e sete componentes da solução energética foram removidos da composição original sem implicações negativas na eficiência de expressão da ErA, simplificando desta forma o processo de SPLC. Em relação ao segundo objetivo, ErA fusionada à SpyCatcher (SpyCatcher_ErA) foi conjugada com êxito em bacteriófagos capazes de expressar SpyTag fusionadas na porção N-terminal das proteínas pIII (SpyTag_pIII) e pVIII (SpyTag_pVIII). A porcentagem de formação dos conjugados entre SpyCatcher_ErA e SpyTag_pIII ((ErA)5-pIII) foi de 6% enquanto formação dos conjugados entre SpyCatcher_ErA e SpyTag_pVIII ((ErA)50-pVIII) foi de 46%, valores estes confirmados por atividade enzimática. Solução contendo conjugados foram injetados em camundongos e sequenciados/titulados com êxito. Não houve diferença de depuração plasmática entre (ErA)5-pIII e bacteriófago controle, mas houve maior taxa de eliminação de (ErA)50-pVIII em relação ao mesmo bacteriófago não conjugado à SpyCatcher_ErA. Os resultados aqui apresentados confirmam ser possível expressar ErA com atividade biológica em sistemas de SPLC. Além disso, o sistema de conjugação da ErA a bacteriófagos aqui desenvolvido foi capaz de monitorar a concentração de ErA presente na circulação em função do tempo, tornando-se uma potencial plataforma de desenvolvimento de novas proteoformas da ErA com características clínicas melhoradas

L-Asparaginase (L-ASNase) from Erwinia chrysanthemi (ErA) is a widely used enzyme for treatment of acute lymphoblastic leukemia (ALL). Although its use as a second-line treatment has provided significant clinical benefits, hypersensitivity reactions and a fast clearance rate are recurring L-ASNase-related problems. In addition, extensive and costly production processes are required for the manufacturing of pure ErA. Based on these drawbacks, this current work proposes (1) the study of the use of a cell-free protein synthesis (CFPS) system as a viable platform for the synthesis of ErA and (2) the conjugation of the protein on bacteriophages as an alternative tool for the isolation and monitoring of ErA clearance. Escherichia coli-derived cell extracts supplemented with a creatine phosphate-based energy solution were used to synthesize ErA in vitro. To conjugate ErA on bacteriophages, the SpyTag/SpyCatcher system was implemented: SpyCatcher was fused to the N-terminus of the ErA while filamentous phage strains M13 and fd were engineered in order to display SpyTag on their pIII and pVIII capsid proteins, respectively. Regarding the first goal, the CFPS system was able to express an active ErA. The protein was expressed in the soluble fraction and there presented a significant higher enzymatic activity compared to the control reaction (7.07 ± 0.68 U/mL vs. 1.83 ± 0.14 U/mL). Time required to obtain the cell extract was reduced from 45 to 26 hours, and seven energy solution reagents were removed from the original solution without compromising the efficiency of ErA expression, thus simplifying the CFPS process. With respect to the second goal, ErA fused to SpyCatcher (SpyCatcher_ErA) was sucessfully conjugated on bacteriophages capable of displaying SpyTag fused to the Nterminus of the pIII (SpyTag_pIII) or pVIII (SpyTag_pVIII) proteins. Percentage of conjugate formation between SpyCatcher_ErA and SpyTag_pIII (ErA)5-pIII was 6% whereas conjugate formation between SpyCatcher_ErA and SpyTag_pVIII (ErA)50-pVIII was 46%, values that were confirmed by enzymatic activity. Sample containing conjugates were injected into mice and sucessfully sequenced/titrated. No clearance differences were observed between (ErA)5- pIII and a control bacteriophage, but a higher clearance rate was observed for (ErA)50-pVIII compared to SpyTag_VIII non conjugated to SpyCatcher_ErA. The results here presented confirm the expression of a biologically active ErA from a CFPS system. Besides, the development of a conjugation system capable of linking ErA to bacteriophages could be used as a means to monitor the ErA concentration in the blood as a function of time and also as a potential platform to be used in the development of novel ErA proteoforms with improved clinical properties

Caracterización de la proteína no capsidal 3D, del virus de la fiebre aftosa y producción de anticuerpos policlonales / Characterization of the non-capsided 3d protein, of the foot-and-mouth disease virus and the production of polyclonal antibodies

Las proteínas no capsidales del virus de la fiebre aftosa se utilizan como marcadoras en la evaluación de animales que han estado en contacto con el virus, a diferencia de los inmunizados, ya que la vacuna no debe tener estas proteínas, por lo tanto los animales no deben presentar anticuerpos contra ellas. El objetivo de esta investigación fue la caracterización de la proteína no capsidal 3D y la producción de anticuerpos policlonales in vivo. La proteína se purificó del cultivo de virus inactivo, por cromatografía de intercambio iónico. La elución de los picos fue sometida a electroforesis uni-bidimensional; demostrándose un alto grado de pureza (>90%) en el pico tres, donde se identifico la proteína 3D, por la técnica de MALDI-TOF y electroespray de trampa iónica. La proteína purificada, se inoculó en cabras y el suero hiperinmune fue precipitado y sometido a cromatografía de afinidad para la obtención de inmunoglobulinas; la reacción inmunitaria se confirmó por medio de inmunodifusión y Western blot. El proceso de purificación demostró ser eficiente y útil para la obtención de anticuerpos específicos, los cuales tendrán utilidad en la elaboración de un ensayo inmunoenzimático que mida la pureza de la vacuna frente al contenido de estas proteínas.

The noncapsid proteins of the foot and mouth disease are used as markers in the evaluation of animals that have been in contact with the virus, to discriminated the immunized animals, because the vaccine should not have these proteins, therefore animals should not present antibodies against them. The aim of this investigation was the characterization of the 3D non-capsid protein and the production of polyclonal antibodies in vivo. The protein was purified from the culture of inactivated virus, by ion exchange chromatography. The elution of the peaks were submit an one-two-dimensional electrophoresis; Demonstrated a high degree of purity (> 90%) in peak three, where the 3D protein was identified, by the MALDI-TOF technique and ion trap electrospray. The purified protein, inoculated in goats and the hyperimmune serum, was precipitate out and submitted to affinity chromatography to obtain immunoglobulins; the immune reaction was confirmed by means of immunodiffusion and Western blot. The purification process proved to be efficient and useful for obtaining specific antibodies, which will be useful in the preparation of an immunoenzymatic assay that measures the purity of the vaccine against to the content of these proteins.

Molecular characterization of feline calicivirus variants from multicat household and public animal shelter in Rio de Janeiro, Brazil

ABSTRACT The aim of this study was to perform the molecular characterization of conserved and variable regions of feline calicivirus capsid genome in order to investigate the molecular diversity of variants in Brazilian cat population. Twenty-six conjunctival samples from cats living in five public short-term animal shelters and three multicat life-long households were analyzed. Fifteen cats had conjunctivitis, three had oral ulceration, eight had respiratory signs (cough, sneeze and nasal discharge) and nine were asymptomatic. Feline calicivirus were isolated in CRFK cells and characterized by reverse transcription PCR target to both conserved and variable regions of open reading frame 2. The amplicons obtained were sequenced. A phylogenetic analysis along with most of the prototypes available in GenBank database and an amino acid analysis were performed. Phylogenetic analysis based on both conserved and variable region revealed two clusters with an aLTR value of 1.00 and 0.98 respectively and the variants from this study belong to feline calicivirus genogroup I. No association between geographical distribution and/or clinical signs and clustering in phylogenetic tree was observed. The variants circulating in public short-term animal shelter demonstrated a high variability because of the relatively rapid turnover of carrier cats constantly introduced of multiple viruses into this location over time.

Structure analysis of capsid protein of Porcine circovirus type 2 from pigs with systemic disease

Abstract Economic losses with high mortality rate associated with Porcine circovirus type 2 (PCV2) is reported worldwide. PCV2 commercial vaccine was introduced in 2006 in U.S. and in 2008 in Brazil. Although PCV2 vaccines have been widely used, cases of PCV2 systemic disease have been reported in the last years. Eleven nursery or fattening pigs suffering from PCV2 systemic disease were selected from eight PCV2-vaccinated farms with historical records of PCV2 systemic disease in Southern Brazil. PCV2 genomes were amplified and sequenced from lymph node samples of selected pigs. The comparison among the ORF2 amino acid sequences of PCV2 isolates revealed three amino acid substitutions in the positions F57I, N178S and A190T, respectively. Using molecular modeling, a structural model for the capsid protein of PCV2 was built. Afterwards, the mutated residues positions were identified in the model. The structural analysis of the mutated residues showed that the external residue 190 is close to an important predicted region for antibodies recognition. Therefore, changes in the viral protein conformation might lead to an inefficient antibody binding and this could be a relevant mechanism underlying the recent vaccine failures observed in swine farms in Brazil.

Development of an indirect ELISA based on recombinant capsid protein to detect antibodies to bovine leukemia virus

Abstract Serological testing and culling infected animals are key management practices aiming eradication of bovine leukemia virus infection. Here, we report the development of an indirect ELISA based on BLV recombinant capsid protein (BLVp24r) to detect anti-BLV antibodies in cattle serum. The BLVp24r was expressed in Escherichia coli and purified by affinity chromatography, and then used to set up the ELISA parameters. The Polysorp ® plate coated with 50 ng of antigen/well and bovine serum diluted 1:100 gave the best results during standardization. Using sera from infected and non-infected cattle we set up the cutoff point at 0.320 (OD450 nm) with a sensitivity of 98.5% and specificity of 100.0%. Then, we tested 1.187 serum samples from dairy (736 samples) and beef cattle (451 samples) with unknown status to BLV. We found that 31.1% (229/736) and 9.5% (43/451) of samples amongst dairy and beef cattle, respectively, had IgGs to BLV. The rate of agreement with a commercial competitive ELISA was 84.3% with a κ value of 0.68. Thus, our BLVp24r iELISA is suitable to detect BLV infected animals and should be a useful tool to control BLV infection in cattle.

First report and molecular characterization of an isolate of papaya ringspot virus (PRSV-W) detected in pumpkin in Cuba / Primeiro relato e caracterização molecular de um isolado de papaya ringspot virus (PRSV-W) detectado em planta de abóbora proveniente de Cuba

In this work, a virus isolate collected from pumpkin plants (Cucurbita pepo L.), showing severe symptoms of mosaic and leaf deformation, grown in Cuba, was analyzed using indicator plants, electron microscopy, and phylogenetic analysis. Plants of pumpkin, cv. Caserta, inoculated with this virus isolate showed mosaic, leaf distortion and blistering symptoms, whereas papaya plants were immune and did not show any symptoms. A transmission electron microscopic examination of leaf dip preparations made from infected pumpkin leaves revealed the presence of elongated and flexuous particles, approximately 780-800 x 12 nm in size. Genomic fragments containing the coat protein (CP) and HC-Pro genes, amplified by specific primers for Papaya ringspot virus, W strain (PRSV-W), showed amino acid identities of both genes higher than 94% when compared to other PRSV-W isolates from America. In the phylogenetic tree, this virus isolate has grouped with other virus isolates from America, Australia, and India and was more distant from the Asian isolates. Taken together, the analyses allow the conclusion that this virus isolate is a W strain of PRSV, detected for the first time in Cuba.

Neste trabalho um isolado viral coletado em planta de abóbora (Cucurbita pepo L), apresentando sintomas severos de mosaico e deformação foliar, proveniente de uma lavoura localizada em Cuba, foi analisado utilizando plantas indicadoras, microscopia eletrônica de transmissão e análise filogenética. Plantas de abóbora cv. Caserta, inoculadas com este isolado do vírus mostrou mosaico, distorção foliar e bolhas, enquanto que as plantas de mamão foram imunes e não apresentaram sintoma. Exame ao microscópio eletrônico de tranmissão de telas preparadas com a técnica leaf dip, empregando o extrato de folhas de abóbora infectadas revelou a presença de partículas alongadas e flexuosas, medindo cerca de 780-800 x 12 nm. A análise de fragmentos genômicos contendo os genes da proteína capsidial (CP) e HC-Pro, amplificados por primers específicos para Papaya ringspot virus, estirpe W (PRSV -W), mostrou identidades de aminoácidos superiores a 94 % quando ambos os genes foram comparados a outros isolados americanos de PRSV -W. Na árvore filogenética, este isolado estudado se agrupou com os isolados de PRSV-W da América, Austrália e Índia, ficando mais distante dos isolados asiáticos. Tomadas em conjunto, as análises permitem concluir que este isolado viral pertence à estirpe W do PRSV, detectada pela primeira vez em Cuba.

Phylogenetic analysis of human group C rotavirus circulating in Brazil reveals a potential unique NSP4 genetic variant and high similarity with Asian strains

Group C rotaviruses (RVC) cause gastroenteritis in humans and animals worldwide, and the evidence for a possible zoonotic role has been recently provided. To gain information on the genetic diversity and relationships between human and animal RVC, we sequenced the VP4, VP7, and NSP4 genes of 12, 19, and 15 human strains, respectively, detected in São Paulo state during historical (1988 and 1993) and recent (2007 and 2008) Brazilian rotavirus surveillance. All RVC strains analyzed in the present study grouped into human genotype (G4-P[2]-E2), and did not show any evidence of animal ancestry. Phylogenetic analysis showed that RVC samples detected in 1988 and 1993 clustered together with strains from distinct continents, indicating that historical RVC strains circulating in São Paulo were closely related to those strains circulating worldwide. All three genes (VP7, VP4 and NSP4) of São Paulo RVC strains isolated in 2007-2008 exhibited close phylogenetic relationship with human RVC strains isolated in China and Japan, suggesting that they are genetically linked, and that a gene flow could be occurring between this Asian countries and Brazil. We identified two distinct clusters in the NSP4 phylogenetic tree. One cluster formed exclusively by human Brazilian strains detected in 1997 and 2003-2004 in Rio de Janeiro, Bahia, and Rio Grande do Sul states (Subgroup II) previously described in a different study, that displayed low sequence identities to other human strains formerly published, and to the Brazilian RVC strains (Subgroup I) characterized in the present study. These data suggests the circulation of two genetic profiles of the NSP4 gene in Brazil. High sequence diversity in NSP4 gene was previously reported in Asia, and additional diversity in NSP4 RVC strains spreading in the world should be expected. More in-depth molecular and epidemiological analysis of human RVC throughout the world will be needed to understand their diversity and clarify their evolution, as well as to develop classifications schemes.

Occurrence and molecular characterization of Cucumber green mottle mosaic virus in cucurbit crops of KPK, Pakistan

Field survey of the cucurbit crops revealed a high incidence of Cucumber green mottle mosaic virus (CGMMV) in Khyber Pakhtunkhwa Province (KPK), Pakistan. Among the seven districts surveyed, average percent incidence of CGMMV was recorded up to 58.1% in district Nowshera, followed by 51.1% in district Charsada, 40.5% in district Swabi and 37.3% in district Mardan. In Swat and Dir districts average incidence CGMMV was recorded upto 31.2% and 29.4%, respectively. Among the different crops highest incidence in plain areas of KPK was recorded in bottle gourd (59.3%) followed by 56.3% in Squash, 54.5% in Pumpkin, 45.5% in Melon, 41.7% in Cucumber and 29.9% in Sponge gourd. In Northern hilly areas highest incidence of CGMMV (52.9%) was observed in pumpkin, followed by 49.6% in bottle gourd, 47.3% in squash, 45.1% in Melon 42.3% in cucumber and 41.6% in sponge gourd. Little variability was observed in the coat protein amino acid sequence identities of CGMMV Pakistan isolate, when compared with other reported isolates.

Expression of enterovirus 71 capsid protein VP1 in Escherichia coli and its clinical application

The VPl gene of enterovirus 71 (EV71) was synthesized, construct a recombinant plasmid pET15b/VP1 and expressed in E. coli BL21. The recombinant VP1 protein could specifically react with EV71-infected patient sera without the cross-reaction with serum antibodies of coxsackievirus A16 (CA16), A4, A5, B3 and B5 as well as echovirus 6. In acute and convalescent phases, IgM and IgG antibodies of 182 serum samples were detected by ELISA with recombinant VP1 protein as a coated antigen. The results showed that the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of IgM antibodies in serum samples for the diagnosis of EV71 infection were 90.1, 98.4, 98.8 and 88.7%, respectively; similarly, those of IgG antibodies in serum samples were 82.4, 89.1, 91.5 and 78.1%, respectively. Five of 80 samples (6.25%) from CA16infected patients were detected positive by ELISA with recombinant VP1 protein in which indicated the cross reactions and 0 of 5 samples from patients infected with other enteroviruses including CA4, CA5, CB3, CB5 and echovirus 6. Therefore, the recombinant VP1 protein of EV7l may provide a theoretical reference for establishing an effective antibody screening of IgM for EV71-infected patients with clinically suspected hand, foot, and mouth disease (HFMD).

The shift from low to high non-structural protein 1 expression in rotavirus-infected MA-104 cells

A hallmark of group/species A rotavirus (RVA) replication in MA-104 cells is the logarithmic increase in viral mRNAs that occurs four-12 h post-infection. Viral protein synthesis typically lags closely behind mRNA synthesis but continues after mRNA levels plateau. However, RVA non-structural protein 1 (NSP1) is present at very low levels throughout viral replication despite showing robust protein synthesis. NSP1 has the contrasting properties of being susceptible to proteasomal degradation, but being stabilised against proteasomal degradation by viral proteins and/or viral mRNAs. We aimed to determine the kinetics of the accumulation and intracellular distribution of NSP1 in MA-104 cells infected with rhesus rotavirus (RRV). NSP1 preferentially localises to the perinuclear region of the cytoplasm of infected cells, forming abundant granules that are heterogeneous in size. Late in infection, large NSP1 granules predominate, coincident with a shift from low to high NSP1 expression levels. Our results indicate that rotavirus NSP1 is a late viral protein in MA-104 cells infected with RRV, presumably as a result of altered protein turnover.

Analysis of real time PCR amplification efficiencies from three genomic region of dengue virus / Análisis de las eficiencias de amplificación por PCR en tiempo real de tres regiones genómicas del virus dengue

Early diagnosis of dengue virus (DENV) infection represents a key factor in preventing clinical complications attributed to the disease. The aim of this study was to evaluate the amplification efficiencies of an in-house quantitative real time-PCR (qPCR) assay of DENV, using the non-structural conserved genomic region protein-5 (NS5) versus two genomic regions usually employed for virus detection, the capsid/pre-membrane region (C-prM) and the 3’-noncoding region (3’NC). One-hundred sixty seven acute phase serum samples from febrile patients were used for validation purposes. Results showed that the three genomic regions had similar amplification profiles and correlation coefficients (0.987-0.999). When isolated viruses were used, the NS5 region had the highest qPCR efficiencies for the four serotypes (98-100%). Amplification from acute serum samples showed that 41.1% (67/167) were positive for the universal assay by at least two of the selected genomic regions. The agreement rates between NS5/C-prM and NS5/3’NC regions were 56.7% and 97%, respectively. Amplification concordance values between C-prM/NS5 and NS5/3’NC regions showed a weak (k= 0.109; CI 95%) and a moderate (k= 0.489; CI 95%) efficiencies in amplification, respectively. Serotyping assay using a singleplex NS5-TaqMan® format was much more sensitive than the C-prM/SYBR Green® I protocol (76%). External evaluation showed a high sensitivity (100%), specificity (78%) and high agreement between the assays. According to the results, the NS5 genomic region provides the best genomic region for optimal detection and typification of DENV in clinical samples.

El diagnóstico precoz de la infección por el virus dengue (DENV) constituye un elemento clave para la prevención de las complicaciones clínicas propias de la enfermedad. El objetivo del estudio fue evaluar la detección de DENV mediante un ensayo cuantitativo de PCR-tiempo real (qPCR), desarrollado localmente, utilizando la región no-estructural-5 (NS5), versus dos regiones tradicionalmente empleadas para la detección del virus, la región cápside/pre-membrana (C-prM), y la región noncodificante-3’ (3’NC). Se recolectaron 167 muestras de suero de pacientes en fase aguda de la enfermedad. Las tres regiones génicas tuvieron perfiles de amplificación/coeficientes de correlación similares (0,987-0,999). Sin embargo, la región NS5 tuvo la eficiencia de amplificación más elevada para los cuatro serotipos (98-100%). Durante el proceso de validación, 41,1% (67/167) de las muestras de suero resultaron positivas para DENV al menos por dos de las regiones genómicas empleadas. Los valores de concordancia entre las regiones NS5/C-prM y NS5/3’NC fueron de 56,7% y 97%, respectivamente. La concordancia fue débil entre las regiones NS5/C-prM (k= 0,109; CI 95%), sin embargo, fue moderada entre las regiones NS5/3’NC (k= 0,489; CI 95%). El ensayo de tipificación uniplex en formato NS5/TaqMan® mostró alta sensibilidad (100%) que el protocolo C-prM/SYBRGreen®-I (76%). La validación externa del ensayo mostró una alta sensibilidad (100%), especificidad (78%) y acuerdo alto entre los ensayos utilizados. De acuerdo a los resultados obtenidos, la región NS5 ofrece la mayor opción para la detección y serotipificación del DENV en muestras clínicas.

Genetic diversity of dengue virus serotypes 1 and 2 in the State of Paraná, Brazil, based on a fragment of the capsid/premembrane junction region / Diversidade genética dos vírus dengue sorotipos 1 e 2, no Estado do Paraná, Brasil, baseada no fragmento da junção do gene capsídeo/pré-membrana

INTRODUCTION: The precise identification of the genetic variants of the dengue virus is important to understand its dispersion and virulence patterns and to identify the strains responsible for epidemic outbreaks. This study investigated the genetic variants of the capsid-premembrane junction region fragment in the dengue virus serotypes 1 and 2 (DENV1-2). METHODS: Samples from 11 municipalities in the State of Paraná, Brazil, were provided by the Central Laboratory of Paraná. They were isolated from the cell culture line C6/36 (Aedes albopictus) and were positive for indirect immunofluorescence. Ribonucleic acid (RNA) extracted from these samples was submitted to the reverse transcription polymerase chain reaction (RT-PCR) and nested PCR. RESULTS: RT-PCR revealed that 4 of the samples were co-infected with both serotypes. The isolated DENV-1 sequences were 95-100% similar to the sequences of other serotype 1 strains deposited in GenBank. Similarly, the isolated DENV-2 sequences were 98-100% similar to other serotype 2 sequences in GenBank. According to our neighbor-joining tree, all strains obtained in this study belonged to genotype V of DENV-1. The DENV-2 strains, by contrast, belonged to the American/Asian genotypes. CONCLUSIONS: The monitoring of circulating strains is an important tool to detect the migration of virus subtypes involved in dengue epidemics.

INTRODUÇÃO:A identificação precisa da variante genética do vírus da dengue é importante para compreender a dispersão, virulência e identificação das cepas responsáveis pelas epidemias. O objetivo da pesquisa foi investigar a variação genética do fragmento da junção do gene capsídeo/pré-membrana dos sorotipos 1 e 2. MÉTODOS: Amostras de onze municípios do Estado Paraná, Brasil, foram cedidas pelo Laboratório Central do Paraná e consistiam em isolados de cultura de células da linhagem C6/36 (Aedes albopictus), positivos para técnica de imunofluorescência indireta. O Ribonucleic acid (RNA) dessas amostras foi extraído, seguido da transcrição reversa, reação em cadeia da polimerase (PCR) e nested PCR. RESULTADOS: Co-infecção por DENV-1 e 2 (virus da dengue 1 e 2) foi observada em quatro pacientes, através da técnica Reverse transcriptase-polymerase chain reaction (RT-PCR). Para o DENV-1 a porcentagem de similaridade variou de 95 a 100% comparando com cepas do Genbank. Para o DENV-2 a porcentagem de similaridade variou de 98 a 100%. De acordo com o cladograma gerado, todas as cepas deste estudo se agruparam no genótipo V para DENV-1. Para o DENV-2 foi encontrada a cepa referente ao genótipo asiático/americano. CONCLUSÕES: O monitoramento das cepas circulantes torna-se uma ferramenta importante na detecção da migração dos subtipos do vírus da dengue envolvidos em epidemias.

Production of L1 protein from different types of HPV in Pichia pastoris using an integrative vector

Human papillomavirus (HPV) infection is the most common sexually transmitted disease in the world and is related to the etiology of cervical cancer. The most common high-risk HPV types are 16 and 18; however, the second most prevalent type in the Midwestern region of Brazil is HPV-33. New vaccine strategies against HPV have shown that virus-like particles (VLP) of the major capsid protein (L1) induce efficient production of antibodies, which confer protection against the same viral type. The methylotrophic yeast Pichia pastoris is an efficient and inexpensive expression system for the production of high levels of heterologous proteins stably using a wild-type gene in combination with an integrative vector. It was recently demonstrated that P. pastoris can produce the HPV-16 L1 protein by using an episomal vector associated with the optimized L1 gene. However, the use of an episomal vector is not appropriate for protein production on an industrial scale. In the present study, the vectors were integrated into the Pichia genome and the results were positive for L1 gene transcription and protein production, both intracellularly and in the extracellular environment. Despite the great potential for expression by the P. pastoris system, our results suggest a low yield of L1 recombinant protein, which, however, does not make this system unworkable. The achievement of stable clones containing the expression cassettes integrated in the genome may permit optimizations that could enable the establishment of a platform for the production of VLP-based vaccines.

High prevalence and low E6 genetic variability of human papillomavirus 58 in women with cervical cancer and precursor lesions in Southeast Mexico

Infection with some genotypes of human papillomavirus (HPV) is the most important risk factor associated with cervical cancer (CC). Throughout the world, HPV type 58 prevalence varies from one region to another; it is higher in women from certain countries in Asia and Latin America, such as China and Mexico. Although intratypic variants have been reported on a few occasions, our knowledge about HPV 58 genetic variation remains limited. Therefore, this work aims to (i) determine the prevalence of HPV type 58 amongst Mexican women with invasive CC or precursor lesions and (ii) identify HPV 58 sequence variants. One hundred and forty five colposcopy clinic patients were studied. Genotyping of HPV 16, 18 and 58 was determined by specific nested PCR and HPV 58 variants were detected by direct sequencing. The general prevalence of HPV was 51.7 percent (75/145). HPV 16 was found in 30.6 percent (23/75) and HPV 58 in 24 percent (18/75) of the patients. HPV 18 was not identified in patients with cervical intraepithelial neoplasia (CIN) grade I; it was only found in those with CIN II, with a prevalence of 6.8 percent (3/44). In patients with CC, the prevalence of HPV 16 and 58 was 78.9 percent. Regarding HPV 58 variants, 94.4 percent of the HPV 58 sequences were identical to the prototype strain, whereas one sample showed changes at a single nucleotide. This study demonstrates a high prevalence of HPV 58 and a low genetic variability of E6 sequences amongst Mexican colposcopy patients.

Expression of a hantavirus N protein and its efficacy as antigen in immune assays

Hantavirus cardiopulmonary syndrome (HCPS) has been recognized as an important public heath problem. Five hantaviruses associated with HCPS are currently known in Brazil: Juquitiba, Araraquara, Laguna Negra-like, Castelo dos Sonhos, and Anajatuba viruses. The laboratory diagnosis of HCPS is routinely carried out by the detection of anti-hantavirus IgM and/or IgG antibodies. The present study describes the expression of the N protein of a hantavirus detected in the blood sample of an HCPS patient. The entire S segment of the virus was amplified and found to be 1858 nucleotides long, with an open reading frame of 1287 nucleotides that encodes a protein of 429 amino acids. The nucleotide sequence described here showed a high identity with the N protein gene of Araraquara virus. The entire N protein was expressed using the vector pET200D and the Escherichia coli BL21 strain. The expression of the recombinant protein was confirmed by the detection of a 52-kDa protein by Western blot using a pool of human sera obtained from HCPS patients, and by specific IgG detection in five serum samples of HCPS patients tested by ELISA. These results suggest that the recombinant N protein could be used as an antigen for the serological screening of hantavirus infection.

Detection of EBV-DNA in serum samples of an immunosuppressed child during a three years follow-up: association of clinical and PCR data with active infection

Vinte e quatro amostras de sangue total e de soro foram colhidas durante seguimento por 36 meses de criança de oito anos de idade, imunodeprimida devido a transplante cardíaco. O paciente apresentou VCA-IgG e IgM positivos e EBNA-IgG negativo aos cinco anos de idade quando foi diagnosticada mononucleose infecciosa. Quatorze meses depois o VCA-IgG e o EBNA-IgG eram positivos e o VCA-IgM negativo. Este padrão sorológico persiste desde aquela época mesmo durante episódios sugestivos de reativação. As amostras de sangue total e de soro foram analisadas pela Reação em Cadeia da Polimerase (PCR) que amplificou fragmento oriundo da gp220 do EBV (detecção de 100 cópias virais). Todas as 24 amostras de sangue total e 12 amostras de soro foram positivas por PCR. Com o objetivo de verificar se a detecção de DNA do EBV em soro estaria associada à reativação da doença, os resultados de PCR foram analisados em relação à necessidade de hospitalização e uso de anti-viral. O teste de Kappa mostrou que existe concordância entre a presença de DNA do EBV em soro e a necessidade de hospitalização e tratamento com anti-virais (valor de 0,750; p < 0,001). Concluímos que a detecção de DNA do EBV em amostras de soro de pacientes imunosuprimidos poderia ser usada como marcador laboratorial de atividade da infecção quando técnicas quantitativas de amplificação não estiverem disponíveis.

Producción y purificación de la proteína Core del virus de la hepatitis C en el sistema de expresión del baculovirus para ensayos biológicos / Hepatitis C virus core protein production and purification in a baculovirus expression system for biological assays

Introducción. La infección por el virus de la hepatitis C (VHC) se caracteriza por la alta frecuencia de infección persistente. La capacidad del VHC para inducir alteraciones en la función de las células del sistema inmune, específicamente en las células dendríticas, parece ser una de las estrategias virales implicada en el establecimiento de la infección persistente. Esta estrategia parece estar mediada en gran parte por una de las proteínas estructurales codificada por el genoma del VHC, la proteína Core, unidad estructural de la cápside viral. Objetivo. Una de las limitantes para la evaluación de las propiedades de Core es la obtención y la purificación de la proteína nativa (191 aa). El objetivo de este estudio fue la producción y la purificación de la proteína Core completa en un sistema eucariote para evaluar las propiedades de la proteína en cultivos de células dendríticas humanas. Resultados. En este estudio se logró la producción de la proteína Core completa en el sistema de expresión de baculovirus y su purificación por la técnica de separación por punto isoeléctrico y electroelución. La pureza de la proteína obtenida se confirmó por Western blot y tinción con plata. Estos análisis mostraron dos bandas únicas correspondientes a las isoformas p23 y p21 de la proteína Core, previamente descritas en la literatura. Conclusiones. La proteína obtenida posee varias de las condiciones de la proteína Core nativa, como peso molecular, isoformas y localización subcelular. Los procedimientos descritos en este artículo son aplicables a proteínas asociadas a membranas producidas en sistemas de expresión eucarióticos

Desarrollo de improntas para el diagnostico del virus epstein-barr por inmunofluorescencia indirecta / Development of slides for Epstein-Barr virus diagnosis by indirect immunofluorescence

El vírus de Epstein-Barr (VEB) es el principal agente oncogénico linfotrópico dentro de la família Herpesviridae y se encuentra mundialmente distribuído. La primoinfección se produce en adultos jovenes y se manifesta como mononucleosis infecciosa. La detección de anticuerpos anti-viral cápside antigen (VCA) indica infección previa o presente com VEB. Además, se observan títulos elevados de anticuerpos anti-VCA en las enfermidades neoplásicas asociadas al VEB como los linfomas, em indivíduois HIV-positivos. El objetivo de este estúdio fue el desarrollo y puesta a punto de improntas de células P3HR1 para la detección serológica del VEB por técnicas de inmunofluorescencia indirecta (IFI). Se estimularon cultivos de células P3HR1 en crecimiento exponencial com phorbol-12-mirystoil-13-acetato y se recolectaron alícuotas a distintos tiempos para realizar improntas. Se realizó uma IFI com cada impronta usando como anticuerpo primário um suero VEB-positivo. Se observo un aumento del 11% em la expresión del VCA a las 40 horas post-estimulación, deyendo al 3.5% a las 48 horas. Estos datos fueron corroborados por ensayo de Western blot com inmunodetección. La precisión intra- e inter-lote de las improntas fue evaluada para anticuerpos IgM e IgG, com sueros probados previamente por equipos para esta determinación disponibles en el mercado para el VEB y com sueros reactivos para otros miembros de la família Herpesviridae. No se obtuvieron resultados falsos-negativos ni falsos-positivos para el VEB ni se observo reactividad cruzada com otros herpesvirus. Las improntas desarrolladas constituyen un instrumento para el diagnóstico de la primoinfección del VEB y la detección serológica de anticuerpos IgG anti-VCA de neoplasias asociadas al VEB.

Analysis of differential selective forces acting on the coat protein (P3) of the plant virus family Luteoviridae

The coat protein (CP) of the family Luteoviridae is directly associated with the success of infection. It participates in various steps of the virus life cycle, such as virion assembly, stability, systemic infection, and transmission. Despite its importance, extensive studies on the molecular evolution of this protein are lacking. In the present study, we investigate the action of differential selective forces on the CP coding region using maximum likelihood methods. We found that the protein is subjected to heterogeneous selective pressures and some sites may be evolving near neutrality. Based on the proposed 3-D model of the CP S-domain, we showed that nearly neutral sites are predominantly located in the region of the protein that faces the interior of the capsid, in close contact with the viral RNA, while highly conserved sites are mainly part of beta-strands, in the protein's major framework.