Reply To: A Hello to Virology
From “Dengue Virions and Antigens in Brain and Serum of Infected Mice” Journal of Virology, Oct. 1970, p. 500-506
https://pubmed.ncbi.nlm.nih.gov/5497897/
Much technical info follows, which I’d heretofore mostly avoided. I won’t go into any astounding detail into technical methodology at this time, but it can be addressed at a later date.
In these studies, they reference “rapidly sedimenting infectious hemagglutinin” antigen (RHA), “slowly sedimenting noninfectious hemagglutinin” antigen (SHA). You can think of hemagglutinin as the “spikes” on certain viruses that attach to red blood cells and allow them entry. The virions (viral particles) compose the “RHA” component.
[Production and purification of virus]
“Suckling mice (1- to 3-day ICR strain) were inoculated intracerebrally with 10^4 plaque-forming units (PFU) of New Guinea “C” dengue-2 virus in the 33rd mouse passage. Brains were harvested from moribund mice 5 days later and homogenized (20%, w/v) in 0.02 M tris(hydroxymethyl)aminomethane (Tris) buffer (pH 8.2). These suspensions were clarified by
the addition of 3 mg of protamine sulfate per ml and centrifugation at 9,000 X g for 1 hr at 4 C.
Antigens were sedimented from the supernatant fluid at 105,000X g for 2.5 hr and resuspended in a volume of buffer effecting a 100-fold concentration.
Density gradient centrifugation- Rate zonal separation of dengue virus antigens was carried out in 27-ml gradients preformed with 5 to 25% (w/v) sucrose inTris buffer. Samples (2 ml) were layered on top of each gradient and centrifuged in a Beckman 25.1 rotor at 63,000 X g for 3 hr at 3 C.
Approximately 30 fractions (1 ml) were collected dropwise through the bottom of the centrifuge tube. Under these conditions as previously described (5), RHA (virion) is found in fractions 4 to 8 near the bottom of the tube, SHA is found in fractions 18 to 22, and SCF is found in fractions 27 to 29. Equilibrium centrifugation in CsCl was accomplished by adjusting samples to approximately 1.23 g/cm3 with a saturated solution of CsCl in Tris buffer.”
After centrifugation of a 5-ml sample at 104,000 X g for 40 hr at 3 C (Beckman SW-39 rotor), fractions were collected as above. Refractive indexes of the CsCl fractions were obtained in a Bauch & Lomb refractometer and converted to density by the method of Ifft et al.”
Electron Microscopy:
“Single-drop samples of appropriate sucrose gradient fractions were placed directly onto grids covered with carbon-coated collodion membranes prewashed in chloroform. Specimens were
allowed to settle for several minutes and the excess was removed by pipette. The grids were inverted on water to remove sucrose, dried, and stained with 1% aqueous uranyl acetate. Micrographs were taken at 58,000 magnification in a Hitachi 1 1B electron microscope.”
There were also assays to count the antigens (viral particles in the case of RHA), and determine their infectiousness:
Antigen Assay:
“Hemagglutinating (HA) antigen were carried out in microtiter plates (Linbro Chemical Co., New Haven, Conn.) by standard methods (2) with goose erythrocytes at pH 6.2. Complement fixation tests were carried out by a microtiter modification of the method described by Kent and Fife (4) utilizing hyperimmune mouse ascitic fluid as a source of excess specific antibody (1). Infectivity titrations were carried out by plaque assay in LLC-MK2 cell cultures.”