RESUMO
In this work, changes in Vero cell cultivation methods have been employed in order to improve cell growth conditions to obtain higher viable cell densities and to increase viral titers. The propagation of the 17DD yellow fever virus (YFV) in Vero cells grown on Cytodex I microcarriers was evaluated in 3-L bioreactor vessels. Prior to the current changes, Vero cells were repeatedly displaying insufficient microcarrier colonization. A modified cultivation process with four changes has resulted in higher cell densities and higher virus titers than previously observed for 17DD YFV.
Assuntos
Reatores Biológicos , Contagem de Células , Técnicas de Cultura de Células , Cultura de Vírus/métodos , Vírus da Febre Amarela/crescimento & desenvolvimento , Animais , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Sobrevivência Celular , Chlorocebus aethiops , Meios de Cultura Livres de Soro , Células Vero , Carga ViralRESUMO
Yellow fever is an acute infectious disease caused by prototype virus of the genus Flavivirus. It is endemic in Africa and South America where it represents a serious public health problem causing epidemics of hemorrhagic fever with mortality rates ranging from 20% to 50%. There is no available antiviral therapy and vaccination is the primary method of disease control. Although the attenuated vaccines for yellow fever show safety and efficacy it became necessary to develop a new yellow fever vaccine due to the occurrence of rare serious adverse events, which include visceral and neurotropic diseases. The new inactivated vaccine should be safer and effective as the existing attenuated one. In the present study, the immunogenicity of an inactivated 17DD vaccine in C57BL/6 mice was evaluated. The yellow fever virus was produced by cultivation of Vero cells in bioreactors, inactivated with ß-propiolactone, and adsorbed to aluminum hydroxide (alum). Mice were inoculated with inactivated 17DD vaccine containing alum adjuvant and followed by intracerebral challenge with 17DD virus. The results showed that animals receiving 3 doses of the inactivated vaccine (2 µg/dose) with alum adjuvant had neutralizing antibody titers above the cut-off of PRNT50 (Plaque Reduction Neutralization Test). In addition, animals immunized with inactivated vaccine showed survival rate of 100% after the challenge as well as animals immunized with commercial attenuated 17DD vaccine.
Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Vacina contra Febre Amarela/imunologia , Febre Amarela/prevenção & controle , Vírus da Febre Amarela/crescimento & desenvolvimento , Animais , Anticorpos Neutralizantes/biossíntese , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Reatores Biológicos/virologia , Chlorocebus aethiops , Desinfetantes/farmacologia , Imunidade Humoral , Esquemas de Imunização , Camundongos Endogâmicos C57BL , Testes de Neutralização , Propiolactona/farmacologia , Análise de Sobrevida , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologia , Células Vero , Cultura de Vírus , Vacina contra Febre Amarela/administração & dosagem , Vírus da Febre Amarela/imunologia , Vírus da Febre Amarela/isolamento & purificação , Vírus da Febre Amarela/patogenicidadeRESUMO
Yellow fever (YF) is an endemic disease in some tropical areas of South America and Africa that presents lethality rate between 20 and 50%. There is no specific treatment and to control this disease a highly effective live-attenuated egg based vaccine is widely used for travelers and residents of areas where YF is endemic. However, recent reports of rare, sometimes fatal, adverse events post-vaccination have raised concerns. In order to increase safety records, alternative strategies should be considered, such as developing a new inactivated vaccine using a cell culture based technology, capable of meeting the demands in cases of epidemic. With this goal, the production of YF virus in Vero cells grown on microcarriers and its subsequent purification by chromatographic techniques was studied. In this work we investigate the capture step of the purification process of the YF virus. At first, virus stability was studied over a wide pH range, showing best results for the alkaline region. Considering this result and the pI of the envelope protein previously determined in silico, a strong anion exchanger was considered most suitable. Due to the easy scalability, simplicity to handle, absence of diffusional limitations and suitability for virus handling of membrane adsorbers, a Q membrane was evaluated. The amount of antigen adsorbed onto the membrane was investigated within the pH range for virus stability, and the best pH for virus adsorption was considered to be 8.5. Finally, studies on gradient and step elution allowed to determine the most adequate salt concentration for washing (0.15M) and virus elution (0.30 M). Under these operating conditions, it was shown that this capture step is quite efficient, showing high product recovery (93.2±30.3%) and efficient DNA clearance (0.9±0.3 ng/dose).
Assuntos
Cultura de Vírus/métodos , Vírus da Febre Amarela/isolamento & purificação , Adsorção , Animais , Chlorocebus aethiops , Cromatografia por Troca Iônica , Concentração de Íons de Hidrogênio , Membranas/química , Células VeroRESUMO
To investigate the genetic characteristics of phosphoprotein (P) and matrix protein (M) genes of variable rabies virus (RV) prevalent in Brazil, the authors genetically characterized the P and M genes from 30 Brazilian RV field isolates. Phylogenetic analysis based on the P and M genes revealed the presence of six RV variants that consisted primarily of three insectivorous bats, the vampire bat, dog and fox in Brazil. Specific amino acid substitutions corresponding to these phylogenetic lineages were observed, with Asp(42) and Glu(62) in the P protein found to be characteristic of Brazilian chiroptera- and carnivora-related RVs, respectively. Amino acid sequence motifs predicted to associate with a viral function in the P and M proteins were conserved among Brazilian RV variants.
Assuntos
Fosfoproteínas/genética , Vírus da Raiva/genética , Proteínas da Matriz Viral/genética , Proteínas Estruturais Virais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Brasil , Variação Genética , Chaperonas Moleculares , Dados de Sequência Molecular , Filogenia , Vírus da Raiva/isolamento & purificaçãoRESUMO
The incidence of canine rabies has been widely reported in Brazil, and new rabies virus (RV) variants, genetically similar to canine RV, have recently been isolated from foxes. In order to derive the epidemiological characteristics of Brazilian Carnivora RV, Brazilian RVs isolated from dogs, cats, and foxes were genetically analyzed. Brazilian Carnivora RV isolates were divided into 2 main lineages. The predominant lineage was found in dogs and cats, which included the Argentinean and Bolivian Carnivora RV isolates, and was extensively distributed throughout Brazil and surrounding countries. The other lineage consisted of three sublineages containing Brazilian dog and fox RV isolates, with the dog sublineages located on an internal branch of 2 fox sublineages, suggesting that RV transmission events might have occurred between foxes and dogs in the past. These results suggest that contact between dogs and wildlife has the potential to generate new rabies variants and that it is important to control RV infection cycles in both dogs and wildlife to prevent spread of rabies infection.