A novel method for purifying bluetongue virus with high purity by co-immunoprecipitation with agarose protein A

Abstract

Background: Bluetongue virus (BTV) is an icosahedral non-enveloped virus within the genus Orbivirus of Reoviridae and exists as 24 distinct serotypes. BTV can infect all ruminant species and causes severe sickness in sheep. Recently, it was reported that BTV can infect some human cancer cells selectively. Because of the important oncolysis of this virus, we developed a novel purifying method for large-scale production. The purifying logic is simple, which is picking out all the components unwanted and the left is what we want. The process can be summarized in 4 steps: centrifugation, pulling down cell debrises and soluble proteins by co-immunoprecipitation with agarose Protein A, dialysis and filtration sterilization after concentration.

Results: The result of transmission electron microscope (TEM) observation showed that the sample of purified virus has a very clear background and the virions still kept intact. The result of 50% tissue culture infective dose (TCID(50)) assay showed that the bioactivity of purified virus is relatively high.

Conclusions: This method can purify BTV-10 with high quality and high biological activity on large-scale production. It also can be used for purifying other BTV serotypes.

Figure 1

Transmission electron microscopy pictures of the unpurified. The sample was a direct collection of the virus culture on Vero cells. Picture A was photographed by digital camera and the virions were marketed by arrows. Picture B was photographed by film and the virions were marketed by arrows. In picture B, an empty shell was observed. Cell debris was also clearly observed in the two pictures.

Figure 2

Transmission electron microscopy pictures of the purified. Picture A was photographed by digital camera and the virions were marketed by arrows. The two-layer structure of BTV virion was perfectly shown in picture A. Picture B was photographed by film and the virions were marketed by arrows. In picture B, an uncoated BTV virion was observed and was marked by arrow.

Figure 3

The activity of purified and unpurified virus. The chart graph shows the process of 50% cells exhibiting CPE as the infected time goes on according to the number of wells. The unpurified virus was used as a criterion to measure whether the purified virus was still active enough. As the infecting days went by, the number of wells appeared 50% CPE was increasing. The curvilinear trend of purified virus is in accordance with unpurified virus, and the wells exhibiting 50% CPE in purified virus per day is slightly less than that of unpurified virus.