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1.
PLoS One ; 8(4): e61790, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23626728

RESUMO

For the rapid production of influenza vaccine antigens in unlimited quantities, a transition from conventional egg-based production to cell-based and recombinant systems is required. The need for higher-yield, lower-cost, and faster production processes is critical to provide adequate supplies of influenza vaccine to counter global pandemic threats. In this study, recombinant hemagglutinin proteins of influenza virus were expressed in the microalga Schizochytrium sp., an established, fermentable organism grown in large scale for the manufacture of polyunsaturated fatty acids for animal and human health applications. Schizochytrium was capable of exporting the full-length membrane-bound proteins in a secreted form suitable for vaccine formulation. One recombinant hemagglutinin (rHA) protein derived from A/Puerto Rico/8/34 (H1N1) influenza virus was evaluated as a vaccine in a murine challenge model. Protective immunity from lethal challenge with homologous virus was elicited by a single dose of 1.7, 5 or 15 µg rHA with or without adjuvant at survival rates between 80-100%. Full protection (100%) was established at all dose levels with or without adjuvant when mice were given a second vaccination. These data demonstrate the potential of Schizochytrium sp. as a platform for the production of recombinant antigens useful for vaccination against influenza.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Microalgas/genética , Infecções por Orthomyxoviridae/prevenção & controle , Estramenópilas/genética , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Relação Dose-Resposta Imunológica , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/administração & dosagem , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Imunização Secundária , Vírus da Influenza A Subtipo H1N1/química , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genética , Influenza Humana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/mortalidade , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia
2.
Virus Genes ; 43(3): 367-75, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21811852

RESUMO

In silico analysis of three Penaeus stylirostris densovirus (PstDNV) promoters, designated P2, P11, and P61, revealed sequence motifs including the TATA box, downstream promoter element (DPE), GC- and A-rich regions, inverted repeat, activation sequence-1 like (ASL) box, and a conserved guanosine (G) at +24. To delineate the regulatory role of these motifs on promoter activity, deletion constructs were made in a promoter assay vector, pGL3 Basic, that contains a luciferase reporter gene. Luciferase assay showed that P2 had the highest promoter activity followed by P11 and P61 in Sf9 cells. The deletions of inverted repeat, DPE, and GC-rich regions in P2 had the highest negative impact on this promoter. Deletions of DPE, G at the +24, and ASL box in P11 had the highest negative impact on this promoter activity. In P61, DPE and G at +24 are the two key regulators of transcriptional activity. Identification of the key transcriptional regulators is important in understanding the PstDNV pathogenesis in shrimp. This information is also valuable in constructing shrimp viral promoter-based vectors for protein expression in insect cell culture system as well as in shrimp.


Assuntos
Densovirus/genética , Penaeidae/virologia , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Densovirus/isolamento & purificação , Dados de Sequência Molecular , TATA Box
3.
J Virol Methods ; 172(1-2): 54-9, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21192980

RESUMO

The propagation of Taura syndrome virus (TSV) in primary hemocyte culture of Pacific white shrimp (Penaeus vannamei) was investigated. Purified TSV was inoculated into a 24 h old primary hemocyte culture and the development of cytopathic effects was monitored. The cell morphology started changing within 6 h post-inoculation; TSV-infected hemocytes started shrinking and granular structures began to form on the cell surface. There was a gradual loss of cell viability and, by 48 h post-inoculation, most cells detached from the bottom of the 96 well microplate. The propagation of TSV during the 48 h time course studied was measured by real-time RT-PCR. TSV copy number reached the highest level by 12 h post-inoculation and then started to decrease. Using an anti-TSV polyclonal antibody, the 55 kDa VP1 capsid protein was detected by Western blot analysis. The data suggest that shrimp primary hemocyte culture supports TSV replication and could be used as a tool for the study of host-virus interactions in TSV pathogenesis.


Assuntos
Dicistroviridae/fisiologia , Penaeidae/virologia , Replicação Viral/fisiologia , Animais , Proteínas do Capsídeo/metabolismo , Sobrevivência Celular , Células Cultivadas , Efeito Citopatogênico Viral , Hemócitos/patologia , Hemócitos/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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