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PubMed1999

Production of reovirus type-1 and type-3 from Vero cells grown on solid and macroporous microcarriers.

Two strains of reovirus were propagated in Vero cells grown in stationary or microcarriers cultures. Vero cells grown as monolayers on T-flasks or in spinner cultures of Cytodex-1 or Cultispher-G microcarriers could be infected with reovirus serotype 1, strain Lang (T1L), and serotype 3, strain Dearing (T3D). A regime of intermittent low speed stirring at reduced culture volume was critical to ensure viral infection of cells in microcarrier cultures. The virus titre increased by 3 to 4 orders of magnitude over a culture period of 150 h. Titres of the T3D reovirus strain were higher (43%) compared to those of the T1L strain in all cultures. Titres were significantly higher in T-flask and Cytodex-1 microcarrier cultures compared to Cultispher-G cultures with respect to either reovirus type. The viral productivity in the microcarrier cultures was dependent upon the multiplicity of infection (MOI) and the cell/bead ratio at the point of infection. A combination of high MOI (5 pfu/cell) and high cell/bead loading (>400 for Cytodex-1 and >1,000 for Cultispher-G) resulted in a low virus productivity per cell. However, at low MOI (0.5 pfu/cell) the virus productivity per cell was significantly higher at high cell/bead loading in cultures of either microcarrier type. The maximum virus titre (8.5 x 10(9) pfu/mL) was obtained in Cytodex-1 cultures with a low MOI (0.5 pfu/cell) and a cell/bead loading of 1,000. The virus productivity per cell in these cultures was 4,000 pfu/cell. The lower viral yield in the Cultispher-G microcarrier cultures is attributed to a decreased accessibility of the entrapped cells to viral infection. The high viral productivity from the Vero cells in Cytodex-1 cultures suggests that this is a suitable system for the development of a vaccine production system for the Reoviridae viruses.

Animals↗
PubMed1999

Gene therapy using SV40-derived vectors: what does the future hold?

Effective genetic therapy requires both a fragment of genetic material to be used therapeutically and a means to deliver it. We began to study simian virus-40 (SV40) as a vector for gene transfer because available gene delivery vehicles did not provide for the full range of therapeutic uses. Other vectors are variably limited by immunogenicity, difficulties in production, restricted specificity, low titers, poor transduction efficiency, etc. In theory recombinant viral vectors based on SV40 (rSV40) should not, on the other hand, be similarly constrained. rSV40 vectors are easily manipulated and produced at very high titer, stable, lacking in immunogenicity, and capable of providing sustained high levels of transgene expression in both resting and dividing cells. The principle limitation of SV40-derived vectors is the size of the packageable insert (</=5 kb). The rationale for developing SV40 as a gene therapy vector is reviewed. Our studies with rSV40 gene transfer have focused mostly on hematopoietic progenitor cells (CD34+) and their derivatives, and on gene delivery to the liver. In both settings, in vitro and in vivo, SV40 has proven to be very effective. It is thus a promising gene delivery vehicle that can complement others currently in use or under development.

Animals↗
PubMed1976

The aetiology of diarrhoea in newborn infants.

Diarrhoea is a common problem in newborn infants in hospital nurseries. In the past, sporadic diarrhoea was often attributed to dietary indiscretion by the mother, and epidemic diarrhoea was though to be caused by an unknown infectious agent. Techniques with which to locate non-cultivable viruses and untypable enteropathogenic strains of Escherichia coli allow reevaluation of the aetiology of diarrhoea in newborn infants. Preliminary results from Melbourne, Australia, suggest that most diarrhoea in newborn infants is induced by a specific infectious agent. During 1975 the agent most often identified from sporadic and epidemic diarrhoea in hospital nurseries was a reovirus-like particle ("duovirus"). Enterotoxin-producing strains of E. coli were rarely isolated. Future attempts to protect newborn infants from developing diarrhoea must be based on an accurate understanding of the aetiology of this disease.

Australia↗
PubMed2003

Virus-like particle production at low multiplicities of infection with the baculovirus insect cell system.

The baculovirus insect cell expression system (BEVS) was used for the production of self-forming Porcine parvovirus-like particles (VLPs) in serum-free medium. A low multiplicity of infection (MOI) strategy was used to overcome an extra virus amplification step, undesirable in industrial production, and to minimize the virus passage effect. It was confirmed that the time of infection (TOI) and MOI are dependent variables. Higher cell densities were obtained at low MOIs, keeping a constant TOI; however, both volumetric and specific productivities were lower. In synchronous infection, at high MOI, the specific productivity decreased when the cells were infected in the late phase of growth. Product degradation due to cell lysis strongly influenced the optimal time of harvest (TOH). Time of harvest was found to be highly dependent on the MOI, and a direct relationship with the cell yield was obtained. Analysis of the culture medium reveals that glutamine depletion occurs in the late phase of the growth. Supplementation of glutamine to uninfected cell cultures resulted in an increased cell yield. Its addition to cultures infected in the middle phase of the growth curve was also able to restore the productivity levels, but addition to cells in their stationary phase caused no observable effect on product expression. The study clearly shows that for a specific TOI it is not obvious what the correct MOI should be to obtain the best volumetric productivity.

Algorithms↗
PubMed2004

Rabies virus production in high vero cell density cultures on macroporous microcarriers.

The purpose of the study was to investigate the rabies virus multiplication in Vero cell cultures performed on porous microcarriers, MCs (cellulose-Cytopore and gelatin-Cultispher G), which provide higher available surface area compared with solid (nonporous) MCs (DEAE-Cytodex 1). In a set of experiments performed at the same MC concentration (MCs per milliliter), cell densities regularly obtained in porous MC cultures were comparable, but almost twice as high as those in solid MC cultures. In addition, 41.1 +/- 3.9-, 35.2 +/- 2-, and 19.6 +/- 5.8-fold increases in cell concentration, relative to the initial cell number, along with maximum rabies virus titers of 6.3 +/- 0.3 x 10(4), 5 +/- 0.1 x 10(4), and 4.3 +/- 0.2 x 10(4) FFD(50)/mL were observed in Cytopore, Cultispher G, and Cytodex 1 MC cultures, respectively. When higher concentrations of MCs were employed, lower performances of virus production and MC-cell occupation (cells per MC or cells per square millimeter) were observed. Cell attachment to MCs was shown to be faster for Cytopore MCs and Cytodex 1 MCs than for Cultispher G MCs. Concerning the kinetics of cell multiplication on MCs, exponential cell growth, at similar specific cell growth rates, took place on Cytopore, Cultispher G, and Cytodex 1 MCs. In addition, cell densities as high as 2.1 +/- 0.2 x 10(6) cells/mL on Cytopore MCs, 1.8 +/- 0.1 x 10(6) cells/mL on Cultispher G MCs, and 1 +/- 0.3 x 10(6) cells/mL on Cytodex 1 MCs were regularly obtained in batch cultures. Optical as well as scanning and transmission electron microscopy studies carried out to analyze MC structure, MC cell occupation, and cell permissivity to virus infection demonstrated that there was uniform cell distribution in the external and internal areas of the MCs, suggesting an efficiency of virus synthesis. Our results indicate the usefulness of these supports for rabies virus antigen production, as well as possibilities for further optimization.

Animals↗
PubMed2004

Mosquito and mammalian cells grown on microcarriers for four-serotype dengue virus production: variations in virus titer, plaque morphology, and replication rate.

Dengue (DEN) viruses consisting of four distinct serotypes cause diseases such as dengue fever, dengue hemorrhagic fever, and dengue shock syndrome in humans. Most of the dengue viruses can be effectively propagated in some mosquito and mammalian cell lines. In this study, we applied microcarrier cell culture technology to study two relevant aspects involving dengue virus, one on biotechnology of cell growth and virus production, and the other on virus biology concerning genetic variation of a virus population. We investigated the growth of C6/36 mosquito cells and Vero cells grown on Cytodex 1 microcarriers. High-titer DEN virus production can be achieved in C6/36 and Vero cells infected at low cell inoculation density, in the lag-phase cell stage, and at low multiplicity of infection (MOI). The maximum titers produced for DEN-1, DEN-3, and DEN-4 viruses were approximately 10- to 10,000-fold lower than for DEN-2 virus produced in C6/36 and Vero cells grown on microcarriers. The DEN-2 virus produced in C6/36 cells displayed far more extensive plaque heterogeneity than in Vero cells. Microcarrier C6/36 mosquito cell culture appeared to be the most effective system for four-serotype DEN virus production. Interestingly, some selected variants of DEN virus may outgrow in Vero cells when using a T-flask culture. These results may provide useful information for DEN vaccine development.

Animals↗
PubMed2005

Mathematical model of influenza A virus production in large-scale microcarrier culture.

A mathematical model that describes the replication of influenza A virus in animal cells in large-scale microcarrier culture is presented. The virus is produced in a two-step process, which begins with the growth of adherent Madin-Darby canine kidney (MDCK) cells. After several washing steps serum-free virus maintenance medium is added, and the cells are infected with equine influenza virus (A/Equi 2 (H3N8), Newmarket 1/93). A time-delayed model is considered that has three state variables: the number of uninfected cells, infected cells, and free virus particles. It is assumed that uninfected cells adsorb the virus added at the time of infection. The infection rate is proportional to the number of uninfected cells and free virions. Depending on multiplicity of infection (MOI), not necessarily all cells are infected by this first step leading to the production of free virions. Newly produced viruses can infect the remaining uninfected cells in a chain reaction. To follow the time course of virus replication, infected cells were stained with fluorescent antibodies. Quantitation of influenza viruses by a hemagglutination assay (HA) enabled the estimation of the total number of new virions produced, which is relevant for the production of inactivated influenza vaccines. It takes about 4-6 h before visibly infected cells can be identified on the microcarriers followed by a strong increase in HA titers after 15-16 h in the medium. Maximum virus yield Vmax was about 1x10(10) virions/mL (2.4 log HA units/100 microL), which corresponds to a burst size ratio of about 18,755 virus particles produced per cell. The model tracks the time course of uninfected and infected cells as well as virus production. It suggests that small variations (<10%) in initial values and specific rates do not have a significant influence on Vmax. The main parameters relevant for the optimization of virus antigen yields are specific virus replication rate and specific cell death rate due to infection. Simulation studies indicate that a mathematical model that neglects the delay between virus infection and the release of new virions gives similar results with respect to overall virus dynamics compared with a time delayed model.

Animals↗
PubMed1980

Concentration of baboon endogenous virus in large-scale production by use of hollow-fiber ultrafiltration technology.

A process is described in which the baboon endogenous virus (BaEV) is produced under optimum conditions in cell culture, and concentrated by hollow-fiber ultrafiltration technology under conditions of large-scale production. This system has advantages over conventional systems in that the flow rate is increased 2.5-fold during concentration. Thermal inactivation of BaEV was retarded by the addition of lactose glutamate to the harvested tissue culture fluid. After concentration, at least 91% of the virus RNA-directed DNA polymerase is recovered with a concomitant increase in infectious virus. Materials needed for modifying described systems may be obtained from commercial sources.

Animals↗
PubMed2003

Human cytomegalovirus impairs dendritic cell function: a novel mechanism of human cytomegalovirus immune escape.

Human cytomegalovirus (HCMV) employs multiple mechanisms to evade the immune system and succeeds to persist lifelong in the host. Human dendritic cells (DC) are the main antigen-presenting cells and play the key role in inducing and maintaining immune responses. Here, we studied the interaction of HCMV with DC. We found that DC, irrespectively of their stage of maturation, were fully permissive for HCMV when endothelial cell-adapted HCMV strains were applied. When fibroblast-adapted strains were used, viral replication was abrogated at the level of immediate early (IE) and/or early (E) gene expression. Irrespective of the HCMV strain used, infection of DC prevented the signal delivery essential for T cell activation in a multistep manner. Furthermore, we observed an altered expression of adhesion molecules. This might contribute to an impairment of DC migration. Our data indicate that a soluble factor induced by IE and/or E genes is involved in these processes. The impairment of DC function upon HCMV infection may contribute to virus-mediated immunosuppression and help the virus to establish persistence in the host.

Adaptation, Physiological↗
PubMed1989

In vitro replication and expression of hepatitis B virus from chronically infected primary chimpanzee hepatocytes.

Primary chimpanzee hepatocytes were maintained in vitro utilizing a serum-free medium. Hepatocyte functions were sustained throughout the culture period as demonstrated by the synthesis and secretion of liver-specific plasma proteins characteristic for differentiated hepatocytes. Hepatocyte cultures established from a chimpanzee chronically infected with human hepatitis B virus exhibited the synthesis and secretion of hepatitis B virus proteins into the medium. In addition, the de novo replication of hepatitis B virus was documented by the recovery of virus, exhibiting an endogenous DNA polymerase activity, from the tissue culture medium. Therefore, both the long-term maintenance of differentiated hepatocytes and the expression of hepatitis B virus from these primary cultures were sustained in the serum-free medium.

Animals↗