Rapid Preparation of Hemagglutinins of Togaviruses from Infected Cell Culture Fluids

Abstract

Antigens in infected cell culture fluids can be easily concentrated by polyethylene glycol precipitation to yield suitable hemagglutinating and complement-fixing antigens for several togaviruses. Although accumulation of hemagglutinin (HA) of Japanese B encephalitis (JBE) virus in infected cell culture fluids has been described (5, 8), little or no HA activity has been recovered for other togaviruses (9, 10). [The term togavirus was recommended by the Subcommittee of Vertebrate Viruses (1) to the International Committee on Nomenclature of Viruses for the arboviruses belonging to serogroups A and B.] We routinely prepare HA of slowgrowing group B and other togaviruses for HAinhibition (HI) tests by concentrating infected culture fluids by using polyethylene glycol (PEG) precipitation (7, 15). Confluent monolayers of PS cells or Vero cells are inoculated with 1/100 infected mouse brain stock for 1 hr, washed, and incubated at 37 C in Eagle's medium or medium 199 supplemented with 0.2% bovine serum albumin. Culture methods, cell lines, and virus strains employed were described previously (6, 13, 14). When cytopathic effect develops, harvested fluids are clarified at 10,000 x g for 10 min at 4 C and then made 8% with respect to PEG 6000 (Koch-Light Ltd., Colnbrook, Bucks, England) by adding 40% (w/v) stock solution at pH 7.8 in Hanks salt solution. After 1 hr at 4 C, virus is sedimented at 10,000 x g for 30 min, the fluid is decanted and drained, and This investigation was supported by a grant from the National Health and Medical Research Council of Australia.

Mutagenesis of the dengue virus type 2 NS3 proteinase and the production of growth-restricted virus

“…Plaques were then counted 7 days after infection. Concentrated stocks of some viruses were produced by precipitation with polyethylene glycol (PEG) (Della-Porta & Westaway, 1972 ). The preparation of rabbit polyclonal antiserum directed against DEN-2 NS3 (residues 355–593) has been described previously (Teo & Wright, 1997 ).…”

Reactions of purified hemagglutinating antigens of flaviviruses with 19S and 7S antibodies

“…Fluids were harvested at 48 and/or 72 h postinfection, except for harvests of Kokobera virus (10 days). The virus yield in pooled fluid harvests was concentrated by precipitaton with 7% polyethylene glycol, and RHA and SHA were separated by sedimentation through 5 to 25% (wt/vol) sucrose density gradients (5,16). MVE virus was labeled with [3H]-leucine during growth in Vero cells.…”

“…Flaviviruses (formerly group B arboviruses) may be readily concentrated and then purified from infected cell culture fluids (or infected mouse brain suspensions) by rate zonal sedimentation, yielding a band of rapidly sedimenting hemagglutinin (RHA) representing the virions, and a slower-sedimenting hemagglutinin (SHA) devoid of ribonucleic acid (5,8,10,16). SHA is a ring-shaped particle 14 nm in diameter (9) comprising the envelope glycoprotein, V-3, the smallest structural protein, V-1, plus in some preparations a nonstructural protein, 8,10).…”

Immune response in rabbits to virion and nonvirion antigens of the Flavivirus kunjin

“…Fla- ' Present address: CSIRO Animal Health Research Laboratory, Parkville, Victoria 3052, Australia. vivirus-infected suckling mouse brain (SMB) or tissue culture fluid contains the virion and other viral antigens separable by rate-zonal sedimentation in sucrose density gradients (5,12,13). There are two peaks of hemagglutinin (HA): the rapidly sedimenting hemagglutinin (RHA) or virion which contains three proteins, V1, V2, and V3, and the slowly sedimenting hemagglutinin (SHA), a nonvirion antigen made up of two of the three virion proteins (V1 and V3) and a virus-specified cytoplasmic protein (NV-2) (12,20).…”

“…There are two peaks of hemagglutinin (HA): the rapidly sedimenting hemagglutinin (RHA) or virion which contains three proteins, V1, V2, and V3, and the slowly sedimenting hemagglutinin (SHA), a nonvirion antigen made up of two of the three virion proteins (V1 and V3) and a virus-specified cytoplasmic protein (NV-2) (12,20). A third antigen found at the top of the gradient is the soluble complement-fixing (SCF) antigen (5,13). All these antigens were shown to possess both group-and type-specific antigenic determinants (2,20).…”