Paraformaldehyde protects of hepatitis C virus particles during ultracentrifugation.
Divergent buoyant densities of hepatitis C virus (HCV) have been reported. If the destruction of HCV particles occurs during the ultracentrifugation process to separate fractions with different densities, an accurate evaluation of the HCV buoyant density is difficult. To examine this concern, changes were examined in HCV RNA titer of each density fraction after paraformaldehyde fixation of virus particles in the sera of 9 patients with chronic HCV infection. Serum was treated with 4% paraformaldehyde, and the density fractions were then separated by ultracentrifugation. The HCV RNA titer of each fraction was determined by reverse transcription-polymerase chain reaction (RT-PCR) assay, and the results were compared with those obtained from the serum without paraformaldehyde fixation. After fixation, the HCV RNA titer was significantly increased in the 1.11-1.14 g/mL fraction (P=0.0018), and decreased in the 1.14-1.17 and 1.17-1.20 g/mL fractions (P=0.0457 and 0.0003, respectively). Using immunogold electron microscopy, it was found that morphologically destroyed HCV particles are present mainly in the 1.17 g/mL fraction of paraformaldehyde-untreated samples, whereas the intact HCV virion particles are present in the 1.12 and 1.14 g/mL fractions of the paraformaldehyde-treated samples. These results suggest that the destruction of HCV virions occurs during the ultracentrifugation process and that paraformaldehyde treatment protects from destruction. It was also considered that the accurate buoyant density of the HCV virion is 1.11-1.14 g/mL. This study describes a useful method for the purification of HCV virions, and provides new insights for elucidating the physicochemical properties of HCV particles.
Fujita
N
N
Third Department of Internal Medicine, Mie University School of Medicine, Mie, Japan. naika-3@clin.medic.mie-u.ac.jp
Kaito
M
M
Ishida
S
S
Nakagawa
N
N
Ikoma
J
J
Adachi
Y
Y
Watanabe
S
S
D000328:Adult; D000368:Aged; D002499:Centrifugation, Density Gradient; D005260:Female; D005404:Fixatives; D005557:Formaldehyde; D016174:Hepacivirus / Q000737:chemistry / Q000302:isolation & purification*; D019698:Hepatitis C, Chronic / Q000821:virology*; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008854:Microscopy, Electron; D008875:Middle Aged; D011108:Polymers; D012367:RNA, Viral / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014461:Ultracentrifugation; D014771:Virion / Q000648:ultrastructure
D005404:Fixatives; D011108:Polymers; D012367:RNA, Viral; D005557:Formaldehyde; C003043:paraform