Tests in mice of a dengue vaccine candidate made of chimeric Junin virus-like particles and conserved dengue virus envelope sequences.

Two new vaccine candidates against dengue virus (DENV) infection were generated by fusing the coding sequences of the self-budding Z protein from Junin virus (Z-JUNV) to those of two cryptic peptides (Z/DENV-P1 and Z/DENV-P2) conserved on the envelope protein of all serotypes of DENV. The capacity of these chimeras to generate virus-like particles (VLPs) and to induce virus-neutralizing antibodies in mice was determined. First, recombinant proteins that displayed reactivity with a Z-JUNV-specific serum by immunofluorescence were detected in HEK-293 cells transfected with each of the two plasmids and VLP formation was also observed by transmission electron microscopy. Next, we determined the presence of antibodies against the envelope peptides of DENV in the sera of immunized C57BL/6 mice. Results showed that those animals that received Z/DENV-P2 DNA coding sequences followed by a boost with DENV-P2 synthetic peptides elicited significant specific antibody titers (≥6.400). Finally, DENV plaque-reduction neutralization tests (PRNT) were performed. Although no significant protective effect was observed when using sera of Z/DENV-P1-immunized animals, antibodies raised against vaccine candidate Z/DENV-P2 (diluted 1:320) were able to reduce in over 50 % the number of viral plaques generated by infectious DENV particles. This reduction was comparable to that of the 4G2 DENV-specific monoclonal cross-reactive (all serotypes) neutralizing antibody. We conclude that Z-JUNV-VLP is a valid carrier to induce antibody-mediated immune responses in mice and that Z/DENV-P2 is not only immunogenic but also protective in vitro against infection of cells with DENV, deserving further studies. On the other side, DENV's fusion peptide-derived chimera Z/DENV-P1 did not display similar protective properties.

Production of lignocellulolytic enzymes from floriculture residues using Pleurotos ostreatus / Producción de enzimas lignocelulolíticas a partir de residuos de floricultura empleando Pleurotus ostreatus / Produçâo de enzimas lignoceluliticas a partir de residuos de floricultura utilizando Pleurotus ostreatus

Floriculture is a vital agro-industrial sector in the Colombian economy; the export of flowers positively impacts employment and the balance of trade. However, this industry could negatively impact the environment if its waste products are not handled properly. These flower residues, rich in lignin, hemicellulose and cellulose, could be a cost-effective raw material to produce enzymes. Here, we evaluate the production of lignocellulolytic enzymes by degradation of Chrysanthemum and Rosa residues using Pleurotus ostreatus, and manganese sulfate and copper sulfate as inductors. From the two residues, we obtained laccase, manganese peroxidase, endoglucanase, exoglucanase, and (3-glucosidase. The use of inductors, favored all enzyme activities except for (3-glucosidase. The enzymes that displayed the highest activity were laccase (4,693.4 U/L and 2,640 U/L from the residues of Chrysanthemum and Rosa, respectively) and (3-glucosidase (9,513 U/L and 6,811.9 U/L). The enzyme that showed the lowest activity was endoglucanase (11.5 U/L and 15.4 U/L). Under the conditions evaluated, the best substrate for enzyme production is Chrysanthemum wastes; the extracts obtained had higher enzymatic activity than the extracts from Rosa residues.

En Colombia la floricultura es un sector agro-industrial importante, con impactos positivos en el empleo y la balanza comercial. Sin embargo, tiene impacto negativo en el medio ambiente porque genera alto volumen de residuos. Estos residuos, ricos en lignina, hemicelulosa y celulosa, podrían ser una materia prima de bajo costo para la producción de enzimas. En este trabajo se estudió en la producción de enzimas lignocelulíticas por la degradación con Pleurotus ostreatus de residuos de Chrysanthemum y Rosa, usando como inductores sulfato de manganeso y de cobre. A partir de ambos residuos se obtuvieron lacasa, manganeso peroxidasa, endoglucanasa, exoglucanasa y p-glucosidasa. Los inductores favorecieron todas las actividades enzimáticas, excepto para p-glucosidasa. Las enzimas que tuvieron mayores actividades fueron lacasa (4,693.4 U/L y 2,640 U/L a partir del residuo de Chrysanthemum y Rosa, respectivamente) y p-glucosidasa (9,513 U/L y 6,811.9 U/L). La enzima que tuvo menor actividad fue endoglucanasa (11.5 U/L y 15.4 U/L). Bajo las condiciones evaluadas, el mejor residuo para producción de enzimas fue Chrysanthemum, porque los extractos tuvieron mayor actividad enzimática que los producidos a partir de Rosa.

Na Colombia, a floricultura è um importante setor da indùstria agrícola, com impactos positivos sobre o sector laboral na balança comercial. Além disto, tem impacto negativo sobre o meio ambiente, pois gera grandes volumes de resíduos. Estes resíduos, com altos conteùdos em lignina, hemicelulose e celulose, podem ser uma matèria-prima de baixo custo para a produçâo de enzimas. Neste traballio foi estudada a produçâo da enzimas lignoceluliticas pela degradaçâo com Pleurotus ostreatus de residuos de Chrysanthemum e Rosa, utilizando como indutores sulfatos de cobre e manganês. A partir destes resíduos foram obtidos lacase, manganês peroxidase, endoglucanase, exoglucanase e β-glicosidase. O uso de indutores favoreceu as atividades enzimáticas, exceto β-glicosidase. As enzimas que apresentaram atividades mais elevadas foram lacase (4,693.4 U/L e 2,640 U/L a partir do Chrysanthemum e Rosa, respetivamente) e β-glicosidase (9,513 U/L e 6,81.9 U/L). A enzima que apresentou menor atividade foi a endoglucanase (11.5 U/L e 15.4 U/L). Nas condiçôes testadas, o melhor resíduo para a produçâo da enzima foi Chrysanthemum, porque os extratos tinham uma atividade enzimática mais elevada que aqueles produzidos a partir de Rosa.

Standardization of an ELISA test using a recombinant nucleoprotein from the Junin virus as the antigen and serological screening for arenavirus among the population of Nova Xavantina, State of Mato Grosso / Padronização de um teste de ELISA utilizando a nucleoproteína recombinante de vírus Junin como antígeno e triagem sorológica para Arenavírus na população de Nova Xavantina, Estado do Mato Grosso

INTRODUCTION: Arenavirus hemorrhagic fever is a severe emerging disease. METHODS: Considering that the levels of antibodies against arenavirus in the Brazilian population are completely unknown, we have standardized an ELISA test for detecting IgG antibodies using a recombinant nucleoprotein from the Junin virus as the antigen. This protein was obtained by inserting the gene of the Junin virus nucleoprotein into the genome of Autographa californica nucleopolyhedrovirus, using the Bac-to-Bac baculovirus expression system. This recombinant baculovirus was used to infect S. frugiperda cells (SF9). RESULTS: The infection resulted in synthesis of high concentrations of recombinant protein. This protein was detected on 12.5 percent polyacrylamide gel and by means of Western blot. Using the standardized ELISA test, 343 samples from the population of Nova Xavantina were analyzed. We observed that 1.4 percent of the serum samples (five samples) presented antibody titers against arenavirus. CONCLUSIONS: These results show the population studied may present exposure to arenavirus infection.

INTRODUÇÃO: A febre hemorrágica por Arenavirus é uma severa doença emergente. MÉTODOS: Considerando que os níveis de anticorpos contra Arenavirus na população brasileira é totalmente desconhecido, nos padronizamos um teste de ELISA para detecção de anticorpos IgG usando uma nucleoproteína recombinante do vírus Junin como antígeno. Esta proteína foi obtida pela inserção do gene da nucleoproteína do vírus Junin no genoma do vírus Autographa californica nucleopolyhedrovirus, utilizando o sistema de expressão em Baculovírus, Bac-To-Bac. Este baculovirus recombinante foi utilizado para infecção de células de S. frugiperda (Sf9). RESULTADOS: A infecção resultou na produção de altas concentrações de proteína recombinante. Esta proteína foi detectada em gel de poliacrilamida 12,5 por cento, e em Western blot. Utilizando o teste de ELISA padronizado, foram analizadas 343 amostras provenientes da população de Nova Xavantina. Observamos que 1,4 por cento dos soros (5 amostras) apresentavam títulos de anticorpos contra arenavírus. CONCLUSÕES: Estes resultados sugerem que a população estudada pode estar sendo exposta a infecções por arenavírus.

Genomic features of attenuated Junín virus vaccine strain candidate.

Junin virus strain Candid #1 was developed as a live attenuated vaccine for Argentine haemorrhagic fever. In this paper, we report the nucleotide sequences of L RNA of Candid #1 and examine the relationship to its more virulent ancestors Junin virus XJ#44 and XJ 13 (prototype) and other closely and distantly related arenaviruses. Comparisons of the nucleotide and amino acid sequences of L and Z genes of Candid #1 and its progenitor strains revealed twelve point mutations in the L polypeptide that are unique to the vaccine strain. These changes could be provisionally associated with the attenuated phenotype. In contrast, Z ORF was completely conserved among all strains.

Molecular cloning and sequence analysis of the Anticarsia gemmatalis multicapsid nuclear polyhedrosis virus GP64 glycoprotein.

The gp64 locus of Anticarsia gemmatalis multicapsid nucleopolyhedrovirus isolate Santa Fe (AgMNPV-SF) was characterised molecularly in our laboratory. To this end, we have located and cloned a AgMNPV-SF genomic DNA fragment containing the gp64 gene and sequenced the complete gp64 locus. Nucleotide sequence analysis indicated that the AgMNPV gp64 gene consists of a 1500 nucleotide open reading frame (ORF), encoding a protein of 499 amino acids. Of the seven gp64 homologues identified to date, the AgMNPV gp64 ORF shared most sequence similarity with the gp64 gene of Orgyia pseudotsugata MNPV. The GP64 from AgMNPV is the smallest baculoviral envelope glycoprotein found to date, differing in 10 or more residues from the other group I nucleopolyhedroviruses. The biological activity of AgMNPV GP64 protein was assessed by cell fusion assays in UFL-AG-286 cells using the obtained recombinant plasmids. In the upstream and downstream regions, relative to the gp64 ORF, we found different conserved transcriptional and post-transcriptional regulatory elements, respectively.