RESUMO
Foot-and-mouth disease is responsible for severe economic losses to the livestock industry of Pakistan. This study aimed to use Swiss albino mice as a cost-effective experimental animal model to study different immunological and histopathological aspects of FMDV instead of natural targeted species like cattle. After isolation of field isolates FMDV on BHK-21 cell line, biological titer of the virus and mice infectious dose50 was calculated. Virus was injected in 45 Swiss albino mice (group A) through intraperitoneal route. The gross, histopathological and immunopathological lesions in heart, trachea and lungs were recorded at different day's intervals. Histopathologically, the heart showed congestion, hemorrhages and necrosis of cardiac muscles. Trachea showed deciliated epithelium and lungs showed hemorrhages, bronchial edema and alveolar emphysema. Immunohistochemical studies revealed the presence of virus in cardiac muscles, tracheal and bronchial epithelium and alveolar lumen. The findings evoked a thought that laboratory animals could be an alternative to large animals to meet budget limitations for further research on foot-and-mouth-disease.
A febre aftosa (FMD) é responsável por graves perdas econômicas para a indústria pecuária do Paquistão. Este estudo teve como objetivo usar camundongos albinos suíços como um modelo animal experimental de baixo custo para estudar diferentes aspectos imunológicos e histopatológicos do FMDV em vez de espécies naturais como o gado. Após o isolamento dos isolados de campo do FMDV na linhagem celular BHK-21, calculou-se o título biológico do vírus e a dose infecciosa dos camundongos50. O vírus foi injetado em 45 camundongos albinos suíços (grupo A) por via intraperitoneal. As lesões macroscópicas, histopatológicas e imunopatológicas no coração, traqueia e pulmões foram registradas em diferentes intervalos de dias. Histopatologicamente, o coração apresentava congestão, hemorragias e necrose dos músculos cardíacos. A traqueia apresentava epitélio deciliado e os pulmões apresentavam hemorragias, edema brônquico e enfisema alveolar. Estudos imuno-histoquímicos revelaram a presença de vírus em músculos cardíacos, epitélio traqueal e orçamentárias para pesquisas adicionais sobre a febre aftosa
Assuntos
Ratos , Vírus da Febre Aftosa/patogenicidade , Febre Aftosa , Animais de Laboratório/anatomia & histologiaRESUMO
The production of biopharmaceuticals as vaccines in serum-free media results in reduced risk of contamination and simpler downstream processing. The production of enveloped viruses and viral vectors such as Semliki Forest Virus (SFV) typically requires lipids that are provided by supplementation with animal serum, so production under serum-free conditions is challenging. In this work, the capacity to deliver genetic material of SFV-viral replicon particles (SFV-VRPs) produced in BHK-21 cells adapted to serum-free medium (BHK/SFM) was evaluated. Three transgenes were evaluated: GFP used as a model protein, while hepatitis C virus nonstructural protein 3 protease domain (HCV-NS3p) and rabies virus glycoprotein (RVGP) were selected based on their distinct nature (enzyme and glycoprotein, respectively). BHK/SFM cells produced a sevenfold higher number of SFV-VRPs, as determined by qRT-PCR. These particles showed similar capacities of infecting BHK/FBS or BHK/SFM cells. GFP expression was evaluated by flow cytometry, HCV-NS3p activity by enzymatic assay, and RVGP expression by ELISA and Western Blot. Expression analysis revealed higher levels of GFP and HCV-NS3p in BHK/SFM, while the levels of RVGP were similar for BHK/SFM and BHK/FBS. In conclusion, the BHK/SFM cells showed increased SFV-VRP production yields, without affecting vector infectivity or heterologous gene expression, hence validating the use of BHK/SFM for industrial applications.
RESUMO
The production of biopharmaceuticals as vaccines in serum-free media results in reduced risk of contamination and simpler downstream processing. The production of enveloped viruses and viral vectors such as Semliki Forest Virus (SFV) typically requires lipids that are provided by supplementation with animal serum, so production under serum-free conditions is challenging. In this work, the capacity to deliver genetic material of SFV-viral replicon particles (SFV-VRPs) produced in BHK-21 cells adapted to serum-free medium (BHK/SFM) was evaluated. Three transgenes were evaluated: GFP used as a model protein, while hepatitis C virus nonstructural protein 3 protease domain (HCV-NS3p) and rabies virus glycoprotein (RVGP) were selected based on their distinct nature (enzyme and glycoprotein, respectively). BHK/SFM cells produced a sevenfold higher number of SFV-VRPs, as determined by qRT-PCR. These particles showed similar capacities of infecting BHK/FBS or BHK/SFM cells. GFP expression was evaluated by flow cytometry, HCV-NS3p activity by enzymatic assay, and RVGP expression by ELISA and Western Blot. Expression analysis revealed higher levels of GFP and HCV-NS3p in BHK/SFM, while the levels of RVGP were similar for BHK/SFM and BHK/FBS. In conclusion, the BHK/SFM cells showed increased SFV-VRP production yields, without affecting vector infectivity or heterologous gene expression, hence validating the use of BHK/SFM for industrial applications.
RESUMO
The recent spread of Zika virus (ZIKV) in the Americas and the Pacific has reached alarming levels in more than 60 countries. However, relatively little is known about the disease on a virological and epidemiological level and its consequences for humans. Accordingly, a large demand for in vitro derived Brazilian ZIKV material to support in vitro and in vivo studies has arisen. However, a prompt supply of ZIKV and ZIKV antigens cannot be guaranteed as the production of this virus typically using Vero or C6/36 cell lines remains challenging. Here we present a production platform based on BHK-21 suspension (BHK-21SUS) cells to propagate Brazilian ZIKV at larger quantities in perfusion bioreactors. Scouting experiments performed in tissue culture flasks using adherent BHK-21 and Vero cells have demonstrated similar permissivity and virus yields for four different Brazilian ZIKV isolates. The cell-specific yield of infectious virus particles varied between respective virus strains (1-48PFU/cell), and the ZIKV isolate from the Brazilian state Pernambuco (ZIKVPE) showed to be a best performing isolate for both cell lines. However, infection studies of BHK-21SUS cells with ZIKVPE in shake flasks resulted in poor virus replication, with a maximum titer of 8.9×103PFU/mL. Additional RT-qPCR measurements of intracellular and extracellular viral RNA levels revealed high viral copy numbers within the cell, but poor virus release. Subsequent cultivation in a perfusion bioreactor using an alternating tangential flow filtration system (ATF) under controlled process conditions enabled cell concentrations of about 1.2×107cells/mL, and virus titers of 3.9×107PFU/mL. However, while the total number of infectious virus particles was increased, the cell-specific yield (3.3PFU/cell) remained lower than determined in adherent cell lines. Nevertheless, the established perfusion process allows to provide large amounts of ZIKV material for research and is a first step towards process development for manufacturing inactivated or live-attenuated ZIKV vaccines.
Assuntos
Técnicas de Cultura de Células/métodos , Cultura de Vírus/métodos , Zika virus/crescimento & desenvolvimento , Animais , Reatores Biológicos , Contagem de Células , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Células Vero , Carga Viral , Vacinas Virais , Replicação Viral , Zika virus/fisiologiaRESUMO
Mammalian cells are the most frequently used hosts for biopharmaceutical proteins manufacturing. Inoculum quality is a key element for establishing an efficient bioconversion process. The main objective in inoculation expansion process is to generate large volume of viable cells in the shortest time. The aim of this paper was to optimize the inoculum preparation stage of baby hamster kidney (BHK)-21 cells for suspension cultures in benchtop bioreactors, by means of a combination of static and agitated culture systems. Critical parameters for static (liquid column height: 5, 10, 15 mm) and agitated (working volume: 35, 50, 65 mL, inoculum volume percentage: 10, 30 % and agitation speed: 25, 60 rpm) cultures were study in T-flask and spinner flask, respectively. The optimal liquid column height was 5 mm for static culture. The maximum viable cell concentration in spinner flask cultures was reached with 50 mL working volume and the inoculum volume percentage was not significant in the range under study (10-30 %) at 25 rpm agitation. Agitation speed at 60 rpm did not change the main kinetic parameters with respect to those observed for 25 rpm. These results allowed for a schedule to produce more than 4 × 10(9) BHK-21 cells from 4 × 10(6) cells in 13 day with 1,051 mL culture medium.
RESUMO
The aim of this work was to study the Polymerase Chain Reaction (PCR) as a tool of quality control of bovine sera and cellular cultures used in the biotechnological industry. A total of 46 samples of bovine sera derived from two slaughterhouses and 33 samples of BHK21 cells derived from two biotechnological industries were evaluated using the primers GPO-3 (sense) and MGSO (antisense). The PCR technique sensibility analysis showed that 280 bp were amplified for the quantities of 50 ng to 0.006 ng of Micoplasma DNA. The primers specificity was confirmed in the test using Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Candida albicans; except by the positive control, none of the samples showed amplification. The presence of Mycoplasma in bovine sera and in the cultures of BHK21 cells showed that 56.5 and 15.2%, respectively, were contaminated. Thus, it was possible to conclude that PCR was a fast and confident technique to detect mycoplasma and that it could be used to control the quality of immunobiological products and inputs, such as sera and cultures of BHK21 cells.
RESUMO
Estudios previos han demostrado que la adaptación de diversos virus a crecer en líneas celulares de vertebrados, conduce a la selección de variantes virales que unen al heparán sulfato (HS) con alta afinidad. En el presente trabajo se determinó la susceptibilidad de cepas del virus dengue (DENV) a la heparina hipersulfatada un análogo al HS, después de pases seriados en células BHK-21. A aislados de campo de los cuatro serotipos de DENV, se les realizaron ocho pases seriados en células BHK-21. La adaptación de los DENV al cultivo celular seleccionó variantes virales con una aumentada capacidad replicativa en células BHK-21 y una incrementada susceptibilidad a la heparina, en relación a las respectivas cepas no adaptadas, obteniéndose una inhibición de la infectividad más significativa en DENV-2 y DENV-4. Las cepas de DENV adaptadas presentaron cambios en la secuencia de aminoácidos de la proteína de envoltura (E), en particular una substitución K204R para DENV-1, N67K para DENV-2, K308R y V452A para DENV-3 y E327G para DENV-4. Estas sustituciones implicaron ganancia de residuos básicos que incrementaron la carga neta positiva de la proteína. Los resultados sugieren, que la adaptación de cepas de DENV a células BHK-21 selecciona variantes virales sensibles a la heparina y que la efectividad de este compuesto varía dependiendo de la cepa viral. Además sugieren que el HS puede jugar un papel importante en la infectividad de las cepas de DENV adaptadas al cultivo celular, a diferencia de los aislados de DENV no adaptados.
Several studies have shown that adaptation of various viruses to grow in certain cell lines of vertebrates, leads to the selection of virus variants that bind heparan sulfate (HS) with high affinity. In this study we investigated the susceptibility of strains of dengue virus (DENV) to oversulfated heparin an analogue of HS after passages in BHK-21 cells. Field isolates of the four serotypes of DENV with a limited number of passes in mosquito cells C6/36HT were serially passaged eight times in BHK-21 cells. The adaptation of the DENV to the cell culture selected viral variants with an increased replicative capacity in BHK-21 cells and an increased susceptibility to heparin compared with the original not adapted strains, with a more significant inhibition of the infectivity in DENV-2 and DENV-4.The E protein of the adapted strains showed changes in the amino acid sequence, particularly at the position K204R to DENV-1, N67K to DENV-2, K308R and V452A for DENV-3 and E327G to DENV-4. These substitutions implicated a gain of basic residues that increased the net positive charge of the protein. These results suggest that adaptation of DENV strains to BHK-21 cells implies changes in the envelope protein, changes associated to the protein reactivity with heparin, the inhibitory effectiveness of this compound varying depending on the viral strain. In addition, these results suggest that the HS can play an important role in the infectivity of the DENV strains adapted to vertebrate cell culture, but not in the infectivity of non-adapted DENV isolates.
Assuntos
Animais , Cricetinae , Vírus da Dengue/efeitos dos fármacos , Heparina/farmacologia , Seleção Genética , Proteínas do Envelope Viral/genética , Aedes/citologia , Linhagem Celular , Chlorocebus aethiops , Vírus da Dengue/crescimento & desenvolvimento , Rim/citologia , Mesocricetus , Modelos Moleculares , Mutação , Mutação de Sentido Incorreto , Ligação Proteica , Conformação Proteica , RNA Viral/genética , Análise de Sequência de RNA , Células Vero , Ensaio de Placa Viral , Cultura de Vírus , Replicação Viral , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/fisiologiaRESUMO
The number of biopharmaceuticals for medical and veterinarian use produced in mammalian cells is increasing year after year. All of them are obtained by stable recombinant cell lines. However, it is recognized that transient gene expression produces high level expression in a short time. In that sense, viral vectors have been extensively used for producing recombinant proteins on lab-scale. Among them, Semliki Forest virus is commonly employed for this purpose. This review discusses the main aspects related to the use of Semliki Forest virus technology as well as its advantages and drawbacks which limit currently its utilization in biopharmaceutical industry on large-scale.
RESUMO
Células animais são alvo de pesquisas visando sua utilização como plataforma para a expressão de proteínas recombinantes, desde vacinas veterinárias até fatores de coagulação para hemofílicos. Exemplos incluem células de inseto Drosophila melanogaster S2 e células de mamífero BHK-21, que vêm sendo estudadas visando a sua utilização para a produção da glicoproteína do vírus da raiva. Independentemente da estratégia de cultivo utilizada, altas concentrações celulares são em geral associadas a uma maior produção da proteína de interesse. O objetivo deste trabalho foi o de investigar estratégias que possibilitariam o cultivo de células animais em altas concentrações celulares. Células de inseto Drosophila melanogaster S2 produtoras da glicoproteína do vírus da raiva foram cultivadas em frascos agitados a 100 rpm e 28, em meio livre de soro SF 900 II suplementado com os aminoácidos asparagina, cisteína, prolina e serina. A adição dos quatro aminoácidos no meio de cultura refletiu em um aumento da concentração celular máxima (XV MÁX) em 16%. Cisteína, quando adicionada isoladamente no meio de cultura, refletiu em uma velocidade específica máxima de crescimento celular (MÁX) 56% maior. Nessa condição, o fator de conversão glicose a célula (YX/GLC) foi 47% maior, indicando um metabolismo de glicose mais eficiente na geração de células. Esses resultados indicam que cisteína é provavelmente substrato limitante do cultivo de células S2AcGPV em meio SF 900 II. [...] Na perfusão, a concentração celular foi cerca de 3 vezes maior do que no cultivo contínuo sem reciclo de células. Provou-se que é possível cultivar células BHK-21 adaptadas a crescimento em suspensão em altas concentrações celulares em escala laboratorial, utilizando biorreator de bancada e spin-filter interno como sistema de retenção celular.
Animal cells have been under research as a platform for the expression of recombinant proteins, ranging from veterinary vaccines to blood coagulation factors for treating hemophilia. Examples include insect Drosophila melanogaster S2 and hamster BHK-21 cells, currently being studied for the production of rabies virus glycoprotein. Regardless of the cultivation strategy, high cell concentrations are usually associated to a higher protein production. Thus, the aim of this research was to investigate animal cell cultivation strategies that would allow higher cell concentrations than those previously reported. Cells of Drosophila melanogaster S2 expressing the rabies virus glycoprotein (S2AcGPV) were cultivated in shake flasks at 100 rpm and 28 , in SF 900 II serum-free medium supplemented with the following amino acids: asparagine, cysteine, proline, and serine. The addition of the four amino acids to the medium increased the maximum cell concentration (XV MAX) in 16%. When only cysteine was added to the medium, the maximum specific growth rate (ÊMAX) was 56% higher. In this condition, the cell yield on glucose (YX/GLC) was 47% higher, indicating a more efficient glucose metabolism. These results show that cysteine is likely a limiting substrate of S2AcGPV cells growing in SF 900 II medium. [...] During perfusion, the cell maintenance coefficient was not negligible and represented 83% of total glucose consumption. These data indicate that the presence of an internal spin-filter may be associated to cell stress. In perfusion, cell concentration was about 3 times higher than that in continuous culture without cell recycle. In conclusion, it was proved that suspension-adapted BHK-21 cells can be cultivated in a laboratory-scale bioreactor with an internal spin-filter, in order to achieve high cell concentrations.
Assuntos
Drosophila melanogaster/fisiologia , Grau de Concentração de Radionuclídeo , Meios de Cultura/classificação , Técnicas de Cultura de Células/métodos , Reatores Biológicos/classificaçãoRESUMO
O vírus da raiva pode estar presente em diferentes tecidos e órgãos, tornandopossível a sua replicação em diversos tipos de culturas celulares. Considerando a fundamental importância da produção desse vírus in vitro para a realização detestes diagnósticos, produção de soros hiperimunes e pesquisas, o objetivo deste estudo foi avaliar a infecção viral das cepas PV (Pasteur Virus) e CVS (Challenge Virus Standard) em linhagem de células BHK-21 (Baby Hamster Kidney). As células foraminfectadas com as cepas PV e CVS e cultivadas em frascos estacionários de cultivo celular e em microplacas com lamínulas, a 37ºC por até 96 horas. A cada três horas foram coletadas alíquotas do sobrenadante dos frascos, para acompanhamento daconcentração das partículas virais, e lamínulas para avaliar a infecção viral nas células. As avaliações foram realizadas por imunofluorescência direta, para definição da maior diluição em que as suspensões virais infectaram 100% da monocamada confluente de células BHK-21 e para avaliar o aumento da intensidade de fluorescência, expressa em cruzes (+ a ++++), identificando o antígeno viral, demonstrado por fotodocumentação. A presença de partículas viraisfoi observada a partir de nove horas pós-infecção, em ambas as cepas. As partículas virais das cepas PV e CVS no sobrenadante foram obtidas a partir de 15 e 18 horas de incubação, respectivamente, sendo observada a maior concentração de partículasnas suspensões virais das duas cepas, 72 horas pós-infecção. Portanto, o protocolo usado demonstrou eficiência, independente da cepa empregada, permitindo a obtenção de bons títulos nas suspensões virais produzidas
Assuntos
Replicação Viral , Vírus da RaivaRESUMO
Cultura de células de linhagem contínua de rim de hamster (BHK-21) demonstrou ser um sistema mais sensível e mais rápido para o isolamento do vírus da caxumba, quando comparado com o de ovos embrionados de galinha. Estas células podem ser cultivadas sem dificuldade, em laboratório, ao contrário das células primárias de rim de macaco, igualmente sensíveis mas que são de difícil obtenção. A utilização de células BHK-21 para o isolamento do vírus da caxumba, do material biológico, constitui uma boa alternativa no diagnóstico das infecções causadas por esse vírus (AU).