Rapid and efficient purification of hepatitis A virus from cell culture.
Hepatitis A virus (HAV) characteristically remains strongly cell-associated when grown in culture, with only small yields in the culture supernatant. Cell factories (6000 cm2) of BS-C-1 cells infected with the cytopathic HM175A.Z strain of HAV for 3, 4 or 7 days were harvested using trypsin to disperse the infected cell monolayer, and cells were collected by low speed centrifugation. More than 70% of the yield of virus and viral antigen can thus be obtained in the packed cell pellet. Packed cell pellets were resuspended in 5 volumes of isotonic buffer and cell membranes lysed by the addition of a non-ionic detergent. After removal of nuclei by centrifugation, ionic detergent was added to the clarified cytoplasmic extract. Under these conditions, HAV particles (virions and empty capsids) are the only particulate material remaining in the sample, and were recovered in a single ultracentrifugation step through discontinuous sucrose/glycerol density gradients. In one day, this method yields viral antigen with minimal cellular contaminants, in a concentrated volume suitable for subsequent biochemical, vaccine or diagnostic uses. The yield of viral antigen over numerous batches varied from 200 to 1600 vaccine-equivalent doses per cell factory, with a titre of up to 1 x 10(10) infectious particles per ml.
Bishop
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NE
Hepatitis Research Unit, Macfarlane Burnet Centre for Medical Research, Victoria, Australia.
Hugo
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DL
Borovec
S V
SV
Anderson
D A
DA
D000818:Animals; D000956:Antigens, Viral / Q000032:analysis / Q000302:isolation & purification*; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004279:DNA, Viral / Q000032:analysis; D006506:Hepatitis A / Q000382:microbiology*; D035921:Hepatitis A Antigens; D006507:Hepatovirus / Q000302:isolation & purification*; D007668:Kidney / Q000166:cytology; D012367:RNA, Viral / Q000032:analysis; D013997:Time Factors; D014764:Viral Proteins / Q000032:analysis; D014773:Virology / Q000379:methods
D000956:Antigens, Viral; D004279:DNA, Viral; D035921:Hepatitis A Antigens; D012367:RNA, Viral; D014764:Viral Proteins