key: cord-267140-vdcf6vok
authors: Trudel, M.; Marchessault, F.; Payment, P.
title: Purification of infectious rubella virus by gel filtration on sepharose 2B compared to gradient centrifugation in sucrose, sodium metrizoate and metrizamide
date: 1981-02-28
journal: Journal of Virological Methods
DOI: 10.1016/0166-0934(81)90032-x
sha: 
doc_id: 267140
cord_uid: vdcf6vok

Abstract Rubella virus was purified by chromatography on Sepharose 2B after concentration by ultrafiltration on hollow fibers and hydroextraction with PEG 20,000. Yields of 40% infectivity and 70% hemagglutinating activity were routinely obtained. Chromatographie purification was compared to ultracentrifugation in sucrose, metrizamide and sodium metrizoate. Yields were lower in sucrose and metrizamide, while sodium metrizoate reduced the infectivity of the virus below detectable levels. These results demonstrate the advantage of Sepharose 2B for the purification of infectious rubella virus.

Density gradient centrifugation in sucrose is applied to the purification of viruses with varying degrees of success (Hinton and Dobrota, 1976) . Iodinated aromatic compounds such as sodium metrizoate or metrizamide have also been suggested for density gradient purification of labile viruses (Hinton and Mullock, 1976) . These compounds are primarily used as X-ray contrast media. Sodium metrizoate is a derivative of triiodo-benzoic acid;

this medium has been used for the separation of cells and microorganisms but is unstable in certain conditions (pH, ionic strength) (Hinton and Dobrota, 1976; Hinton and Mullock, 1976) . A recently introduced X-ray contrast medium, metrizamide (2-[3-acetamido-5-N-methylacetamido-2,4,6-triiodobenza~do]-2-deoxy-D-glucose) is a completely covalent molecule and, as such, forms stable density solutions up to 1.45 g/ml. These solutions are more mobile than equivalent solutions of sucrose or Ficoll. Metrizamide and related compounds should prove useful in situations where more conventional media give poor results (Hinton and Dobrota, 1976) .

The method of choice for the purification of rubella virus has always been sucrose density gradient (Vaheri et al., 1969; Liebhaber and Gross, 1972; Payment et al., 1975; Trudel and Payment, 1980) . Infectious titer yields for purification by one cycle isopycnic banding has been in the range of 15%. Further purification on a second gradient reduces the yield to less than 1% (Vaheri et al., 1969) . In earlier studies, salt gradients (potassium tartrate or citrate, cesium chloride or sulfate) were tried, but also showed problems with regard to recovery of infectivity.

On the other hand, exclusion chromatography of rubella virus on Sephadex G-200 has been reported (Veronelli and Maassab, 1965; Schmidt and Lennette, 1966) and

Sepharose 2B gel filtration has been used successfully for the purification of other viruses (Bengtsson and Philipson, 1964) . More recently, Sepharose gel filtration has been applied to the purification of hepatitis A (Hornbeck et al., 1975/76; Locarnini et al., 1978) poliovirus (Van Wezel et al., 1978) and coronaviruses (Ignatov et al., 1979) with efficiencies up to 85%. This report describes the application of Sepharose 2B gel fitration for the purification of infectious rubella virus in comparison to centrifugation on sucrose, metrizamide and sodium metrizoate.

Rubella virus, strain M-33 (ATCC VR-315) was produced on monolayers of Vero cells grown in Corbeil Bellco TM system (Bellco Glass Co., Vineland, NJ) (Corbeil et al., 1979) . Cells were cultivated in one TM5 module in equal parts of Medium 199 (Hanks' base) and minimal essential medium (Earle's base) supplemented with 4% kaolin-treated calf serum (treated to remove non-specific inhibitors of hemagglutination) and 50 pg/ ml of gentamicin.

Two liters of harvested supernatant containing 16 HAUlO.025 ml (hemagglutinating units) were concentrated by hollow fiber ultrafiltration on an Amicon DH-4 model system (Amicon, MA). The ultrafiltration system was modified with quick-connect fittings and air filters to minimize aerosol dispersion. The concentrator was equipped with an Hl-100 hollow fiber cartridge (molecular weight cut-off of 100,000) and operated according to manufacturer specifications. Minimal volume after DH-4 concentration is 75 ml and concentration efficiency between 75% and 90% (Trudel and Payment, 1980) . The concentrate was separated in aliquots of 5 ml and stored at -70°C.

Ultracentifigation. Concentrated rubella virus was disaggregated by adding 10 mM EDTA and the suspension was clarified by centrifugation at 15,000 g for 20 min at 4°C. Aliquots of 5 ml of viruses were purified by two cycles of density gradient centrifugation in sucrose, metrizoic acid (sodium salt) or metrizamide (Sigma Chemical Co., St. Louis, MO). The virus was layered on a discontinuous 30%/50% (5 ml/5 ml) gradient and centrifuged at 35,000 r.p.m. in a SW-40 rotor for 3 h at 4°C. The virus-containing . The plates were incubated at 4°C for 1 h. One unit of antigen was defined as the highest dilution that produced a complete hemagglutination.

Infectivity titer. Virus preparations were titrated by interference with Echo 11 virus on primary cercopithecus monkey kidney cells (Furesz et al., 1969) .

Protein. Protein concentration was determined using the Bio-Rad protein assay procedure (Bio-Rad Laboratories, CA).

Fractions, positive by hemagglutination, were examined after negative staining with phosphotungstic acid 3%, pH 6, with a Phillips EM 300 microscope.

Osmolulity. Osmolality was measured with an Osmette (Precision Systems, Inc., MA).

Infectious tissue culture fluids were concentrated by hollow fiber ultrafiltration with an efficiency of 76.8% as previously reported (Trudel and Payment, 1980) . Aliquots were on Sepharose 2B with NTE buffer. We first studied the stability of rubella virus infectivity in these media. As can be seen from Table 1 , after 2 h there is an average inactivation of 40-50% in NTE buffer, sucrose and metrizamide. In sodium metrizoate inactivation reached 95%. After 24 h, virus inactivation was higher in NTE buffer and metrizamide than in sucrose. In sodium metrizoate inactivation was nearly complete.

We also measured the osmolality of the solutions used for separation, reasoning that physiological osmolality should prove more suitable for viral purification. The osmolality of NTE buffer and tissue culture medium is within physiological range (260-320 mOsm) while metrizamide is slightly higher (458 mOsm). Both sucrose and sodium metrizoate are over 2000 mOsm which was the limit of our measuring osmometer.

Density gradient centrifugation of rubella virus on sucrose, metrizamide or sodium a Virus (5 ml, 65,566 HAU and 109*6TCID,,) was purified by ultracentrifugation or chromatography as described in Material and methods.

b Sucrose, metr~z~ide and sodium metrizoate osmolality was measured on 40% (w/w) solutions. c All solutions were prepared in NTE buffer (280 mOsm). d NTE buffer was used as eluate for chromatography purification of rubella virus. NA: not applicable.

metrizoate yielded similar isopycnic patterns with similar buoyant densities at 1.19 g/ cm3 ( Table 2) . Recovery of hemagglutinating activity was equivalent for sucrose and metrizamide (62%) and much lower for sodium metrizoate. Infectivity yields'paralleled these findings, except that infectivity was below detectable level with sodium metrizoate.

on Sepharose 2B gave better recovery of hemagglutinating activity (70%) and infectivity (40%) ( Table 2 , Fig. 1 ) than ultracentrifugation.

Purified virus was recovered in eight fractions (fractions 31-38) that corresponded to one of the protein peaks (Fig. 1) . Three protein peaks are resolved (fractions 1) 20-26, 2) 31-38 and 3) 46662).

Peak 1 consisted of cellular debris of over 40 X lo6 molecular weight that Electron microscopic examination of viruses, purified by the four methods, revealed good structural preservation by chromatography (Fig. 2D) , sucrose ( Fig. 2A) , and metrizamide (Fig. ZC) , while sodium metrizoate purified viruses (Fig. 2B ) revealed penetrated virions and less defined hemagglut~ins at the membrane surface. Viruses purified in metrizamide and sucrose were about one-third smaller than those purified by chromatography and had a more relaxed viral configuration especially visible on viruses purified in metrizamide (Fig. 2B) . Viruses purified by chiomatography on Sepharose 2B (Fig. 2B ) showed a more compact structure and a denser hemagglutinin border. Pleomorphic forms were also more evident than by centrifugation.

of rubella virus by multi-step ultracentrifugation has been difficult to achieve because of the fragility of rubella virus. Yields of infectious virions of 0.l-15% have been reported (Vaheri et al., 1969) . Our study reports similar yields (16%). These results indicate the fragility of rubella virus to repeated sucrose gradient centrifugation.

On the other hand, iodinated density gradient media have been recommended for the purification of labile viruses because of their low osmolality (Hinton and MuIlock, 1976) . Purification of rubella virus yielded similar results in sucrose and metrizamide but the infectivity was reduced to undetectable levels in sodium metrizoate. For sodium metrizoate, this was expected as it inactivates the virus infectivity (Table l) , although hemaglutinating activity is not affected. Similar results have been reported for herpes simplex virus, where lo-30% of infectivity was recovered by met&amide gradient centrifugation, while infectivity was not detected after sodium metrizoate purification (Blomberg et al., 1976) .

Exclusion chromatography of rubella virus on Sephadex G-200 has been reported to permit recovery of total infectivity (VeronelIi and Maassab, 1965; Schmidt and Lennette, 1966 ) but did not permit separation of virus from protein and particles of 600,000 molecular weight. Improved separation has been mentioned by Vaheri reportedly using Sepharose 4B, but no results were published. Sepharose 4B (Phannacia Fine Chemicals) has a fractionation range of 6 X 104-20 X lo6 which does not permit separation because the virus migrates with the void volume. Sepharose 2B, with its exclusion limit of 40 X lo6 molecular weight permits fractionation of the virus which is slowed by the Sepharose beads and is separated from the void volume contaminants.

Pearl agar chromatography has been successfully applied to the purification of viruses with recoveries of 50-82% (Bengtsson and Philipson, 1964) . Sepharose 2B has been used to purify hepatitis A virus with yields of 80% (Locarnini et al., 1978) . In our hands, chromatography of rubella virus on Sepharose 2B yields recovery of 70% of the virusassociated hemagglutinin and 40% of the infectivity in peak material. This is by far better than what has been reported so far for the purification of rubella virus. Furthermore, total hemagglutinin recovery is 95%100% when taking in account fractions 42--50

where free hemagglutinin is eluted (Fig. 1) . Chromatographic purification of rubella virus is routinely used in the laboratory and variations between experiments are within a normal range.

Laboratory Techniques in Biochemistry and Molecular biology

The Use of Iodinated Density Gradient Media for Biological separation

This work was jointly supported by a grant from the National Research Council of Canada (Rubella 601) and the Medical Research Council of Canada (MA6682).