Physical and chemical characterization of purified group A arboviruses
Creators: name: Dolana, Gary Harward
Publisher: name: University of Utah
Year: 2012
Subjects: Arboviruses
Gel Absorption

description: It was shown that four group A arboviruses, Western equine encephalitis (WEE), Sindbis, Eastern equine encephalitis (EEE), and Venezuelan equine encephalitis (VEE), have the same buoyant densities in cesium chloride,  Infectivity lost during density gradient centrifugation in cesium chloride was attributed to the combined action of high speed centrifugation and high salt concentration.  The concentration of WEE, Sindbis, EEE9 and VEE viruses was accomplished by using an aqueous two-phase polymer system prepared from sodium, dextran sulfate (NaDS) and polyethylene glycol.  Concentration factors of 53 to 99 and yields of 63 to 94 percent were obtained.  Precipitation of the NaDS from the virus containing bottom phase with 3M KCl reduced yields considerably with two of the four viruses. Virus concentrated in the small bottom phase appeared to be bound to NaDS which remained after KC1precipitation.  The NaDS could not be separated from the virus by centrifugation through polysucrose density gradients nor by column chromatography using DEAE Sephadex.  The purification and concentration of WEE„ Sindbis, and TEE viruses were accomplished using a multi-step procedure„  The virus particles present in fluids from infected chick embryo cell culture monolayers were precipitated by the addition of an equal volume of saturated ammonium sulfate buffered with IM Tris-HCl. The precipitated virus which had been resuspended in 0.01M Tris-HCl buffer pH  7.3 with 0,001M EDTA was absorbed onto magnesium pyrophosphate gel.   The virus eluted from the gel by 0.3M potassium was centrifuged through a sucrose gradient to separate virus from contaminating protein.  Micro-diffusion tests using virus from the sucrose gradient showed a single precipitin band against rabbit antisera.  No activity was observed against rabbit anticalf serum which would detect 5 ug/ml bovine serum albumin.  Virus yields of 20 to 80 per cent were obtained with purification factors of 700 to 1000.  Photodynamic action on the infectivity and hemagglutinating properties of the viruses was investigated.  It was found that 1 x 10[-6] to 7.5 x 10[-7] gm/ml methylene blue chloride provided maximum rates of inactivation of virus infectivity»  The inactivation curves indicated first-order inactivation reactions,,  Plots of reaction rate constants for each virus at pH values between 6.0 and 8.0 showed a pH dependency which appeared to be characteristic for each virus.  The hemagglutinating titers of infective VEE and WEE virus preparations increased upon exposure to visible light in the presence of methylene blue and upon continued exposure the titer decreased rapidly,,  The maximum increase in hemagglutinating titer was observed after all infectivity was lost.  Hemagglutinating antigens prepared by Tween 80-ether and acetone-ether did not increase in titer due to photodynamic action.  The hemagglutinin decreased at a steady rate until no activity was detectable„  Photodynamically inactivated preparations which did not have any demonstrable infectivity or hemagglutinin and inactivated Tween 80-ether antigen were antigenic when injected into rabbits. The density of WEE virus hemagglutinin increased upon exposure to light in the presence of methylene blue and after continued radiation a broad relatively flat peak was observed suggesting the presence of a heterogeneous population of hemagglutinating particles.