Scanning electron microscopy-based approach to understand the mechanism underlying the adhesion of dengue viruses on ceramic hydroxyapatite columns.

Although ceramic hydroxyapatite (HAp) chromatography has been used as an alternative method ultracentrifugation for the production of vaccines, the mechanism of virus separation is still obscure. In order to begin to understand the mechanisms of virus separation, HAp surfaces were observed by scanning electron microscopy after chromatography with dengue viruses. When these processes were performed without elution and with a 10-207 mM sodium phosphate buffer gradient elution, dengue viruses that were adsorbed to HAp were disproportionately located in the columns. However, when eluted with a 10-600 mM sodium phosphate buffer gradient, few viruses were observed on the HAp surface. After incubating the dengue viruses that were adsorbed on HAp beads at 37°C and 2°C, the sphericity of the dengue viruses were reduced with an increase in incubation temperature. These results suggested that dengue virus was adsorbed to the HAp surface by electronic interactions and could be eluted by high-salt concentration buffers, which are commonly used in protein purification. Furthermore, virus fusion was thought to occur with increasing temperature, which implied that virus-HAp adhesion was similar to virus-cell adhesion

Sequential two-step chromatographic purification of infectious poliovirus using ceramic fluoroapatite and ceramic hydroxyapatite columns.

Infectious virus purification techniques are important for vaccine development and gene therapy applications. However, the standardized one-step purification technique using ceramic hydroxyapatite (CHAp) has proven unsuitable for poliovirus. Therefore, we designed a sequential two-step chromatographic technique for purification of the infectious Sabin type 2 vaccine strain of poliovirus from the cell culture supernatant. In the first step, we removed protein contaminants from the Sabin type 2 virus fraction by pH gradient elution on a ceramic fluoroapatite column. In the second step, we removed double-stranded DNA derived from host cells by diluting the virus fraction, directly loading it on a CHAp column, and purifying it using a phosphate gradient with 1 M sodium chloride. This process achieved removal rates of more than 99.95% and 99.99% for proteins and double-stranded DNA, respectively, and was highly reproducible and scalable. Furthermore, it is likely that it will be applicable to other virus species

Observation of HAp surfaces that were incubated under 3 different conditions.

<p>Column D: no incubation; Column E: incubated at 2°C for 7 days; Column F: incubated at 37°C for 7 days. HAp particles of the column head were observed with scanning electron microscopy. The white triangles indicate spherical-shaped viruses. The black triangles indicate flat-shaped viruses.</p

SEM images of control HAp surfaces. A: Untreated HAp particles; B: HAp particles after loading of cell culture fluid (virus-free).

<p>SEM images of control HAp surfaces. A: Untreated HAp particles; B: HAp particles after loading of cell culture fluid (virus-free).</p

The average number of dengue viruses that were observed and counted separately as spherical viruses and flat viruses on the SEM images.

<p>Standard deviation in brackets.</p

Illustration of experimental methods.

<p>A: The flow direction and the division of the hydroxyapatite (HAp) column into 3 parts. After removing the HAp particles from the column, HAp particles were collected in each part of the column. Column head: lower position of the column (upstream side); Mid-column: middle position of the column; Column end: upper position of the column (downstream side). B: Virus elution and incubation processes of HAp columns. Detailed conditions are described in the Materials and methods, section 3–6.</p

The average number of dengue viruses that were observed in the SEM images.

<p>Standard deviation in brackets.</p