RESUMO
Retroviral pseudotypes are broadly used as safe instruments to mimic the structure and surface of highly pathogenic viruses. They have been employed for the discovery of new drugs, as diagnostic tools in vaccine studies, and part of serological assays. Because of their widespread use in research and their potential as tools for quality control, it is important to know their shelf life, stability, and best storage conditions. In this study, we produced pseudotypes carrying the lacZ reporter gene and the hemagglutinin (HA) of avian influenza virus subtypes H5 and H7 to investigate their stability under various storage conditions. We produced pseudotypes with titers of approximately 106 RLU/mL, which decreased to 105-106 RLU/mL after short-term storage at 4 °C (up to 4 weeks). Stability was maintained after long-term storage at -20 °C (up to 12 months), even under storage variations such as freeze-thaw cycles. We conclude that, although the titers decreased by 1 log10 under the different storage conditions, the remaining titers can be readily applicable in many techniques, such as neutralization assays. These findings show that large quantities of retroviral pseudotypes can be safely stored for short- or long-term use, allowing standardization and reduced variation in assays involving retroviral pseudotypes.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A/genética , Lentivirus/fisiologia , Temperatura Baixa , Vírus Defeituosos/genética , Vírus Defeituosos/fisiologia , Genes Reporter/genética , Vetores Genéticos/genética , Vetores Genéticos/fisiologia , Células HEK293 , Humanos , Lentivirus/genéticaRESUMO
ELISA in situ can be used to titrate hepatitis A virus (HAV) particles and real-time polymerase chain reaction (RT-PCR) has been shown to be a fast method to quantify the HAV genome. Precise quantification of viral concentration is necessary to distinguish between infectious and non-infectious particles. The purpose of this study was to compare cell culture and RT-PCR quantification results and determine whether HAV genome quantification can be correlated with infectivity. For this purpose, three stocks of undiluted, five-fold diluted and 10-fold diluted HAV were prepared to inoculate cells in a 96-well plate. Monolayers were then incubated for seven, 10 and 14 days and the correlation between the ELISA in situ and RT-PCR results was evaluated. At 10 days post-incubation, the highest viral load was observed in all stocks of HAV via RT-PCR (10(5) copies/mL) (p = 0.0002), while ELISA revealed the highest quantity of particles after 14 days (optical density = 0.24, p < 0.001). At seven days post-infection, there was a significant statistical correlation between the results of the two methods, indicating equivalents titres of particles and HAV genome during this period of infection. The results reported here indicate that the duration of growth of HAV in cell culture must be taken into account to correlate genome quantification with infectivity.
Assuntos
Vírus Defeituosos/fisiologia , Ensaio de Imunoadsorção Enzimática/métodos , Vírus da Hepatite A/fisiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Replicação Viral/fisiologia , Animais , Linhagem Celular , Macaca mulatta , Fatores de Tempo , Carga Viral , Ensaio de Placa ViralRESUMO
ELISA in situ can be used to titrate hepatitis A virus (HAV) particles and real-time polymerase chain reaction (RT-PCR) has been shown to be a fast method to quantify the HAV genome. Precise quantification of viral concentration is necessary to distinguish between infectious and non-infectious particles. The purpose of this study was to compare cell culture and RT-PCR quantification results and determine whether HAV genome quantification can be correlated with infectivity. For this purpose, three stocks of undiluted, five-fold diluted and 10-fold diluted HAV were prepared to inoculate cells in a 96-well plate. Monolayers were then incubated for seven, 10 and 14 days and the correlation between the ELISA in situ and RT-PCR results was evaluated. At 10 days post-incubation, the highest viral load was observed in all stocks of HAV via RT-PCR (10(5) copies/mL) (p = 0.0002), while ELISA revealed the highest quantity of particles after 14 days (optical density = 0.24, p < 0.001). At seven days post-infection, there was a significant statistical correlation between the results of the two methods, indicating equivalents titres of particles and HAV genome during this period of infection. The results reported here indicate that the duration of growth of HAV in cell culture must be taken into account to correlate genome quantification with infectivity.
Assuntos
Animais , Vírus Defeituosos/fisiologia , Ensaio de Imunoadsorção Enzimática/métodos , Vírus da Hepatite A/fisiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Replicação Viral/fisiologia , Linhagem Celular , Macaca mulatta , Fatores de Tempo , Carga Viral , Ensaio de Placa ViralRESUMO
Modified Vaccinia virus Ankara (MVA) constitutes a good candidate for the development of non-replicative expression viral vectors because it does not replicate in most of mammalian cells. It is essential, for the production of recombinant MVA, the availability of transfer vectors which allow the introduction of desired genes into non-essential regions for in vitro viral replication, by homologous recombination with the viral genome. In the present work, the transfer vectors named VT-MHA and VT-MTK were designed and obtained. They carried genomic regions corresponding to 1-303 and 608-948 positions of the MVA165R gene and 1-244 and 325-534 of the MVA086R gene, respectively, which flank a multiple cloning site for the insertion of foreign genes. In these vectors, the cassettes for the expression of lac Z or uidA genes were cloned, and the activity of the marker enzymes beta-galactosidase and beta-glucuronidase was confirmed in situ. Furthermore, the vector named VT-MTK-GUS was used to obtain and isolate pure recombinant MVA, which carried and expressed the uid A gene. The results herein constitute the basic tools for establishing the methodology to obtain recombinant MVA with the purpose of locally developing non-replicative viral vectors as candidate vaccines.
Assuntos
Vírus Defeituosos/genética , Vetores Genéticos/genética , Vaccinia virus/genética , Animais , Embrião de Galinha , Clonagem Molecular/métodos , DNA Recombinante/genética , Vírus Defeituosos/fisiologia , Genes Reporter , Genes Virais , Vaccinia virus/fisiologia , Replicação ViralRESUMO
A replicon vaccine vector system was developed from an attenuated strain of Venezuelan equine encephalitis virus (VEE). The replicon RNA consists of the cis-acting 5' and 3' ends of the VEE genome, the complete nonstructural protein gene region, and the subgenomic 26S promoter. The genes encoding the VEE structural proteins were replaced with the influenza virus hemagglutinin (HA) or the Lassa virus nucleocapsid (N) gene, and upon transfection into eukaryotic cells by electroporation, these replicon RNAs directed the efficient, high-level synthesis of the HA or N proteins. For packaging of replicon RNAs into VEE replicon particles (VRP), the VEE capsid and glycoproteins were supplied in trans by expression from helper RNA(s) coelectroporated with the replicon. A number of different helper constructs, expressing the VEE structural proteins from a single or two separate helper RNAs, were derived from attenuated VEE strains Regeneration of infectious virus was not detected when replicons were packaged using a bipartite helper system encoding the VEE capsid protein and glycoproteins on two separate RNAs. Subcutaneous immunization of BALB/c mice with VRP expressing the influenza HA or Lassa virus N gene (HA-VRP or N-VRP, respectively) induced antibody responses to the expressed protein. After two inoculations of HA-VRP, complete protection against intranasal challenge with influenza was observed. Furthermore, sequential immunization of mice with two inoculations of N-VRP prior to two inoculations of HA-VRP induced an immune response to both HA and N equivalent to immunization with either VRP construct alone. Protection against influenza challenge was unaffected by previous N-VRP immunization. Therefore, the VEE replicon system was characterized by high-level expression of heterologous genes in cultured cells, little or no regeneration of plaque-forming virus particles, the capability for sequential immunization to multiple pathogens in the same host, and induction of protective immunity against a mucosal pathogen.
Assuntos
Proteínas do Capsídeo , Capsídeo/imunologia , Vírus Defeituosos/fisiologia , Vírus da Encefalite Equina Venezuelana/genética , Vetores Genéticos/genética , Vírus Auxiliares/fisiologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A/imunologia , Vírus Lassa/imunologia , Replicon , Vacinas Combinadas/genética , Vacinas Sintéticas/genética , Vacinas Virais/genética , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos , Capsídeo/biossíntese , Capsídeo/genética , Linhagem Celular , Embrião de Galinha , Chlorocebus aethiops , Cricetinae , Patos , Vírus da Encefalite Equina Venezuelana/imunologia , Vírus da Encefalite Equina Venezuelana/fisiologia , Fibroblastos , Regulação Viral da Expressão Gênica , Glicoproteínas de Hemaglutininação de Vírus da Influenza/biossíntese , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A/genética , Vírus Lassa/genética , Mesocricetus , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/prevenção & controle , RNA/genética , RNA Viral/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Vacinação , Vacinas Atenuadas/imunologia , Vacinas Combinadas/imunologia , Vacinas Sintéticas/imunologia , Células Vero , Proteínas Estruturais Virais/biossíntese , Proteínas Estruturais Virais/genética , Vacinas Virais/imunologiaRESUMO
SIVsm chronically infected cultures were obtained after infection of CEMX174 cells with either SIVsmH3 or SIVsmE660. These phenotypically CD4 cells, formed syncytia but only when cocultivated with CD4+ cells. Single cell clones were derived from these cultures and examined for the production of virus-specific proteins. The majority of the clones expressed SIV p27 antigen and low levels of virus reverse transcriptase activity. Western blot analysis, performed with either monoclonal or polyclonal sera, showed that a chronically infected clone (B7) produced particles which contained envelope (gp135 and gp43), gag precursors and gag proteins (p27, p16 and p8). However, these particles (SIVsmB7) lacked detectable levels of vpx and of integrase, and contained several fusion proteins which expressed viral protease antigens. This defective virus failed to infect established CD4+ cell lines, as well as primary cultures of macrophages and of peripheral blood lymphocytes, obtained both from humans and from rhesus macaques. Lack of infection correlated with lack of viral DNA detection by PCR amplification of genomic DNA extracted from these cell cultures. In addition, SIVsmB7 virus lacked infectivity in vivo. Rhesus macaques inoculated with high concentrations of SIVsmB7 showed no viremia and their PBMC were PCR negative. Thus, B7 cells produced stable, non-infectious virus mutants, which contained env and gag proteins, but lacked detectable amounts of vpx and of enzymes required for virus replication. Due to the high constitutive expression of this virus-like particle, we are now testing this preparation as a vaccine.
Assuntos
Linfócitos T CD4-Positivos/virologia , Células Clonais/virologia , Vírus Defeituosos/fisiologia , Corpos de Inclusão Viral/fisiologia , Vírus da Imunodeficiência Símia/fisiologia , Animais , Sequência de Bases , Fusão Celular , Efeito Citopatogênico Viral , DNA Viral/análise , Vírus Defeituosos/genética , Vírus Defeituosos/isolamento & purificação , Produtos do Gene gag/análise , Genes Virais , Humanos , Macaca mulatta/virologia , Macrófagos/virologia , Microscopia Eletrônica , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Provírus/genética , Provírus/isolamento & purificação , Sequências Repetitivas de Ácido Nucleico , Proteínas dos Retroviridae/biossíntese , Proteínas dos Retroviridae/genética , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/isolamento & purificação , Replicação ViralRESUMO
The human immunodeficiency virus (HIV) is the etiological agent of acquired immunodeficiency syndrome (AIDS). HIV exhibits extensive genetic diversity and it is apparent that an infected individual contains different populations of distinct viral strains, a large proportion of which has been found surprisingly to be defective for replication. A similar phenomenon has also been observed with some cell lines that are known to produce infectious viral particles but harbor defective proviral genomes. Here, we investigated the molecular basis of this phenomenon by cloning proviral genomes of HIV from a cell line that was capable of producing high titers of biologically active HIV particles that readily induced syncytia with CD4+ cell lines and peripheral blood lymphocytes. This cell line was found to contain five proviral genomes, all of which, when tested individually, failed to produce replication-competent viruses upon transfection into human cells. However, when a specific combination of two proviral genomes was used in such transfection studies, it was possible to obtain biologically active, replication-competent viral particles that infected and replicated in CD4+ cell lines and induced syncytia characteristic of HIV. Such a result may be due to homologous recombination between proviral DNAs occurring in cells after transfection and/or complementation of replication-defective proviral DNAs. The diploid nature of the viral RNA genome present in the viral particle may enable the persistence of defective HIV genomes.
Assuntos
Vírus Defeituosos/fisiologia , Genes Virais , Repetição Terminal Longa de HIV , HIV-1/fisiologia , Provírus/fisiologia , Replicação Viral , Linhagem Celular , Clonagem Molecular , DNA Viral/genética , DNA Viral/isolamento & purificação , Vírus Defeituosos/genética , Biblioteca Gênica , HIV-1/genética , Humanos , Provírus/genética , Mapeamento por Restrição , Rabdomiossarcoma , TransfecçãoRESUMO
Infection of Vero cells with Tacaribe virus stocks containing a high ratio of standard (plaque-forming) viruses to defective interfering particles (DIP) induced inhibition of the host cell Ca2+ ATPase (Ca2+ pump) and the ouabain-sensitive Na+/K+ ATPase (Na+/K+ pump). The Mg2+ ATPase which is not involved in cation transport was not affected. The presence of DIP in the inocula protected the cells from alteration of the transport-associated ATPases induced by standard viruses.